The estimation of utilizable amino acids (uAA) of feeds for ruminants using an in vitro incubation...

8/15/2019 The estimation of utilizable amino acids (uAA) of feeds for ruminants using an in vitro incubation technique

1/11


2/11

in vitro incubation technique. They suggested that the developed in vitro incubationtechnique can be used for quick and accurate estimation of uCP content of feeds. As theuCP contains non-protein nitrogen, e.g. in the form of nucleic acid-N it will be more usefulto modify the in vitro incubation technique to estimate the utilizable amino acids (uAA) atthe duodenum for ruminants. As ruminally fistulated sheep are more commonly used thanruminally fistulated cows in many laboratories, in particular as the donor of rumen fluidfor the Hohenheim gas-test (Naumann and Bassler, 2001), the objective of the presentexperiments was to study the feasibility of estimating uAA of different feeds with anin vitro incubation technique, and to compare the uAA estimated with rumen fluid from acow fed with hay with that from a sheep fed with a ration like that used when rumen fluidfor the gas-test is needed.

Materials and methods

Expt. 1: Incubation with rumen fluid from a dairy cow

Animal and feeding

A non-lactating German Friesian cow (live weight 680 kg) fitted with a large rumen fistula(inner diameter 100 mm) was used as the donor of rumen fluid. The animal was fedwith 4 kg grass hay and 50 g mineral mixture twice daily [total dry matter intake (DMI): 7.1kg/day] and had free access to drinking water.

Feed samples

Twenty-four air-dry feeds (Table 3) and six feed mixtures (Table 1) were ground to pass a3-mm sieve for incubation and determination of dry matter. All feedstuffs had been

Table 1. Composition of feed mixtures (%)

Feed mixture

Ingredients 1 2 3 4 5 6 7 8 9 10 11 12

Barley – – – – – – 30 – – – – –Beet pulp (dried) – – 40 40 50 – – – – – – –Citrus pulp – 30 – – – – – 18.4 18.4 21.6 21.6 80Coconut cake – – 30 – – – – – – – – –Corn – 10 – – 50 – – – – – –Corn gluten feed 20 – 20 – – – – – – – – –Corn silage B – – – – – – – 50 50 51 51 –Corn starch – – – – – – – 4.1 4.1 4.9 4.9 18Faba beans 10 – – – – 50 – – – – – –Manioc – 20 – 10 – – 16.5 – – – – –Palm kernel cake – 40 – – – – – – – – – –Protected rapeseed

meal130 – – – – – – – – – – –

Rapeseed meal 1 – – – 30 – – 30 – – – – –Rapeseed meal 2 – – – – – – – 27 – – – –Rapeseed meal 3 – – – – – – – – 27 – – –Soya bean hulls – – 10 – – – 20 – – – – –Soya bean meal 1 – – – – – – – – – 22 – –Soya bean meal 2 – – – – – – – – – – 22 –Mineral mixture – – – – – – 3.5 0.5 0.5 0.5 0.5 2Wheat grain 40 – – 20 – 50 – – – – – –

1Formaldehyde treated

247The in vitro incubation technique for the estimation of uAA


3/11

included in the in vivo trials used for the formulation of equations for prediction of uCP(Lebzien et al., 1996) before.

Incubation

The incubation procedures were based on the in vitro incubation technique of Zhao andLebzien (2000). The rumen fluid was taken just before morning feeding and was filteredthrough one layer of cheese-cloth. Centrifugation tubes that were about 2 cm in diameter,6 cm in length and 80 ml in volume were used as the incubation vessels. About 0.5 g of feed

sample was weighed into each centrifugation tube. The tubes were pre-warmed to 38 Cbefore incubation. The buffers for the incubation solution were made as following:

Table 2. Amino acid (AA) composition of feeds

Crudeprotein

TotalAA

(16 AA)Methionine Lysine Leucine

a a a b a b a b

FeedsBarley 107 105 1.9 1.8 4.2 4.0 8.4 8.0Beet pulp 67 62 1.4 2.3 4.5 7.3 5.2 8.4Citrus pulp 59 46 0.7 1.5 1.9 4.2 3.8 8.3Coconut meal 191 166 2.7 1.6 5.1 3.1 13.3 8.0Corn (n ¼ 2) 107 110 2.0 1.8 3.7 3.4 14.1 12.8Corn gluten feed 201 193 3.9 2.0 8.5 4.4 20.7 10.7Corn silage (n ¼ 2) 85 59 1.2 2.0 1.8 3.0 7.4 12.5Faba bean 258 245 2.2 0.9 16.9 6.9 20.8 8.5Grass meal 109 100 1.6 1.6 4.2 4.2 8.7 8.7Grass silage 127 101 2.0 2.0 3.7 3.7 9.4 9.3Hay (n ¼ 2) 135 100 1.7 1.7 4.7 4.7 8.8 8.8

Manioc 41 34 0.6 1.8 1.9 5.7 2.9 8.4Oats 107 99 1.9 1.9 4.7 4.7 8.3 8.4Palm kernel cake 171 150 3.6 2.4 6.2 4.1 11.4 7.6Protected rapeseed

meal1353 326 7.5 2.3 17.9 5.5 26.7 8.2

Rapeseed meal 2 365 353 8.1 2.3 21.9 6.2 28.6 8.1Rapeseed meal 3 343 332 7.3 2.2 21.6 6.5 26.9 8.1Soya bean hulls 251 217 3.3 1.5 15.8 7.3 17.8 8.2Soya bean meal 1 466 494 6.9 1.4 32.6 6.6 40.5 8.2Soya bean meal 2 427 437 6.1 1.4 28.8 6.6 36.7 8.4Wheat grain 137 130 2.3 1.8 4.8 3.7 10.3 7.9Wheat straw 53 37 0.6 1.6 1.9 5.2 3.1 8.5

Feed mixtures

Mix 1 243 213 4.3 2.0 10.7 5.0 18.3 8.6Mix 2 103 91 2.0 2.2 3.7 4.1 7.6 8.4Mix 3 148 135 2.4 1.8 6.6 4.9 12.0 8.9Mix 4 173 157 3.5 2.2 9.4 6.0 12.7 8.1Mix 5 79 82 1.5 1.8 3.4 4.1 6.6 8.0Mix 6 214 183 3.7 2.0 11.3 6.2 14.8 8.1Mix 7 179 183 3.7 2.0 11.2 6.1 11.1 6.1Mix 8 158 139 3.1 2.2 7.4 5.3 12.6 9.1Mix 9 149 133 2.8 2.1 7.2 5.4 12.1 9.1Mix 10 165 154 2.5 1.6 8.6 5.6 14.0 9.1Mix 11 157 141 2.3 1.6 7.8 5.5 13.1 9.3Mix 12 65 62 1.1 1.8 2.4 3.9 5.5 8.8

a: g/kg dry matter, b: percentage of 16 analysed amino acids (TAA). 1 Formaldehyde treated

248 G. Y. Zhao and P. Lebzien


4/11

Buffer I: 23.5 g Na2HPO4 12H2O (per analysis – p.a.), 12.5 g NaHCO3 (p.a.) and 11.5 gNH4HCO3(p.a.) were dissolved in 400 ml distilled water. NH4HCO3 was used as thenitrogen source for the microbes to replace part of the NaHCO3 in the original buffer(Tilley and Terry, 1963).Buffer II: 23.5 g NaCl (p.a.), 28.5 g KCl (p.a.), 6.0 g MgCl 2 6H2O (p.a.) and 2.63 gCaCl2 2H2O (p.a.), were dissolved in 1000 ml distilled water.

In a beaker, 400 ml of buffer I, 50 ml of buffer II and an adequate volume of distilledwater were mixed to yield a final volume of 500 ml. Two hundred and fifty ml of mixedbuffer and 1000 ml of distilled water were mixed and pre-warmed to 38 C in a flask andthe mixture was continuously gassed with CO2 and was stirred automatically by a

magnetic stirrer. Then 312.5 ml of rumen fluid was added into the flask. After the bufferand rumen fluid were mixed well, 50 ml of buffer-rumen fluid mixture was given into each

Table 3. The utilizable (u) crude protein (CP)-, uAmino acids (AA)-, uMethionine-, uLysine- anduLeucine-content (g/kg incubated dry matter) of feeds and feed mixtures estimated using in vitro

incubation technique with rumen fluid of a cow (Expt. 1)

uMethionine uLysine uLeucineuAA uCP

Regressivecalculated

g/kg (%)2 g/kg (%)2 g/kg (%)2 (g/kg) (g/kg) uCP1 (g/kg)

FeedsBarley 3.9 2.2 14.5 8.1 14.3 8.0 179 164 162Beet pulp 3.2 2.1 12.7 8.2 12.9 8.3 155 157 141Corn 1 3.6 2.2 10.6 6.5 18.6 11.3 164 154 157Citrus pulp 2.2 2.1 9.4 8.9 8.5 8.0 106 136 142Coconut cake 3.5 1.8 7.4 3.9 15.2 8.0 191 236 201Corn gluten feed 3.7 2.1 12.3 7.1 17.0 9.8 174 210 182Corn silage 1 1.7 2.0 5.5 6.4 8.9 10.3 86 105 130Faba bean 2.6 1.6 12.4 7.5 14.4 8.7 166 187 194Grass meal 2.5 2.2 7.8 6.8 10.2 8.9 114 178 137Grass silage 2.9 2.1 9.2 6.8 12.3 9.1 135 165 143

Hay 1 3.0 2.2 10.4 7.6 11.3 8.2 137 139 145Manioc 1.7 2.2 7.4 9.4 6.2 7.8 79 101 131Oat 2.7 2.1 9.2 7.0 10.7 8.2 131 164 140Palm kernel cake 3.7 2.1 7.6 4.4 13.2 7.6 173 163 175Protected rape

seed meal36.4 2.1 18.3 6.0 25.8 8.5 305 343 341

Rapeseed meal 1 5.6 2.1 18.2 6.9 23.8 9.0 263 230 264Rapeseed meal 2 5.1 2.2 15.4 6.7 20.6 8.9 231 256 219Rapeseed meal 3 4.5 2.2 15.2 7.5 18.3 9.0 203 233 214Soya bean hulls 3.4 1.8 14.8 7.6 16.6 8.6 194 207 184Soya bean meal 1 4.5 1.8 17.9 7.1 23.0 9.2 251 289 319Soya bean meal 2 5.0 1.6 21.0 6.6 28.4 9.0 316 309 303Soya bean meal 3 5.0 1.9 19.0 7.3 23.6 9.1 259 270 308Wheat grain 4.0 2.3 15.2 8.6 13.8 7.8 177 173 172Wheat straw 1.1 2.3 3.7 7.7 4.0 8.3 48 63 76

Feed mixturesMix 1 5.3 2.2 16.5 6.8 20.5 8.4 244 258 227Mix 2 3.2 2.2 9.0 6.1 11.9 8.1 147 165 154Mix 3 4.8 2.2 14.9 6.7 18.8 8.4 223 242 172Mix 4 3.2 2.2 13.1 9.0 12.1 8.3 146 161 183Mix 5 2.2 1.9 9.0 7.6 9.5 8.1 118 127 149Mix 7 3.8 2.2 13.8 7.9 14.0 8.0 175 208 177

1Calculated uCP according to: uCP ¼ (187:7 115:4 UDP/CP) DOM + 1.03 UDP/DM2Percentage of 16 analysed amino acids3Formaldehyde treated

249The in vitro incubation technique for the estimation of uAA


5/11

incubation tube and sealed with a rubber stopper fitted with a small glass tube with arubber balloon at the top. Four sets of incubation with two duplicates for each feed sampleand two blanks in each set were used. The incubation tubes were kept at 38 C in a waterbath and were shaken occasionally during incubation. After 24 h of incubation, all tubeswere taken out of the water bath. The pH value was immediately measured. All theincubation residues were freeze-dried for further analysis.

Expt. 2: Incubation with rumen fluid from a sheep

Animal and feeding

An adult sheep (live weight 51 kg), fitted with a large rumen fistula (inner diameter 40 mm),fed with a mixed ration twice daily in two equal meals, was used as the donor of rumenfluid. The mixed ration which had a dry matter content of 85.5% contained 700 g grass hayand 600 g mixed concentrates (65% wheat grain, 29% soya bean meal, 1% soya bean oiland 5% mineral and vitamin mixture). Crude protein content in the ration DM was about

16.5%. The sheep had free access to drinking water.

Feed samples

Twenty-two air-dry feeds (Table 4) and 11 feed mixtures (Table 1), which were used indifferent in vivo experiments before, were ground to pass a 3-mm sieve for incubation anddetermination of dry matter.

Incubation

The rumen fluid was taken 7 h after morning feeding. The amount of NH 4HCO3 in bufferI was reduced to avoid too high levels of ammonia in incubation, and the amount of

NaHCO3 was increased to keep the same level of HCO3 in buffer I as in Expt. 1. Thecomposition of modified buffer I was: 23.5 g Na2HPO4 12H2O (p.a.), 15.0 g NaHCO3(p.a.) and 9.5 g NH4HCO3 (p.a.), dissolved in 400 ml distilled water. The other incubationprocedures and buffer II were the same as in Expt. 1.

Chemical analysis

The feed samples were dried for 6 h at 105 C for the determination of dry matter content.Ammonia N in the incubation residues was analysed by steam distillation and titrationprior to freeze-drying of the residues. Amino acids in the feed samples and the incubationresidues were determined by Degussa (Germany) using an amino acid analyser with cation

exchange resin column and ninhydrin detection according to the EC Directive 98/64/EC of 3 September 1998. Cyst(e)ine and methionine were oxidized to cysteic acid and methioninesulphone respectively prior to hydrolysis. Tryptophan was not determined. In thefollowing only values for the most important amino acids methionine, lysine and leucinewill be listed, to reduce the number of tables and figures.

Calculation and statistical analysis

The uAA and uCP were calculated as following:

uAA ¼ ðAAresidue AAblankÞ=Incubated sample DM ð1Þ

where uAA (g/kg DM) refers to uAA in feed, AA residue (g) refers to AA content of

incubation residue, AAblank (g) to AA content of incubation residue of the blank and DM(kg) to dry matter,



6/11

uCP ¼ ðCPresidue CPammoniaÞ ðCPblankresidue CPblankammoniaÞ½ =

Incubated sample DM ð2Þ

where uCP (g/kg DM) refers to uCP in feed, CPresidue (g) refers to crude protein inincubation residue, CPammonia (g) to crude protein from ammonia in incubation residue,

CPblankresidue (g) refers to crude protein content of residue in blank, CP blankammonia (g)to crude protein from ammonia in incubation residues of blank and DM (kg) to drymatter.

The uCP and uAA determined with in vitro incubation technique were comparedstatistically with the uCP values calculated by the following multiple regression equationbased on 335 in vivo experiments on fistulated cows (Lebzien et al., 1996):

uCP ¼ ð187:7 115:4UDP=CPÞDOM þ 1:03UDP=DM ð3Þ

r 2 ¼ 0:91, CV (%) ¼ 8.9, where uCP (g) refers to utilizable crude protein in feed, UDP (g)refers to rumen undegradable crude protein and DOM (kg) to digestible organic matter.

Results and discussionThe crude protein content and amino acid composition of feeds and feed mixtures are givenin Table 2.

The uCP- and uAA-content of feeds and feed mixtures

The in vitro determined uCP and uAA as well as utilizable (u) Methionine, uLysine anduLeucine in the incubation residues per kg incubated DM of Expt. 1 are shown in Table 3.

On an average the in vitro determined uCP values were only 5 g or 2.6% higher than thevalues calculated by regression. But seven out of the 30 values differ by more than 30 g or15%. That is quite high, but the coefficient of variation for the prediction equation (3) used

for the calculation of the uCP content, amounted also to 8.9% ( Lebzien et al., 1996) andthe differences can be caused as well by the inherent error of the prediction equation asindependent reference as by errors of the in vitro method.

It was found that there was a close relationship between regressive calculated uCP (X ,g/kg) and in vitro determined uCP (Y , g/kg): Y ¼ 0:93X þ 17:50; r 2 ¼ 0:84; p


7/11

uMethionine (r 2 ¼ 0:90; p


8/11

similar to that of Zhao and Lebzien(2000). But although r 2 is nearly the same as in Expt. 1,the relationship differs more from Y ¼ X than in Expt. 1 and points to a systematic effect.The same was true for the relationship between the regressive calculated uCP and thein vitro determined uAA (Fig. 2).

It was also observed that there is a close relationship between the in vitro determineduCP and uAA ðr 2 ¼ 0:96; p


9/11

percentages of duodenal protein (consisting of undegraded dietary protein and rumenmicrobial protein) are 0.6–3.0, 4.7–9.5 and 6.8–11.9%, respectively. Clark et al. (1992)reported that the ranges of methionine, lysine and leucine percentages of rumen microbialprotein are 1.1–4.9, 4.9–9.5 and 5.3–9.7% respectively. The means and ranges forpercentages of methionine, lysine and leucine in TAA of incubation residues (Tables 3 and4), microbial protein and duodenal digesta protein are compared in Table 5. The resultsindicate that the contents of methionine, lysine and leucine in TAA of incubation residuesare within or quite close to the ranges of methionine, lysine and leucine of duodenalprotein from in vivo experiments and the ranges of rumen microbial protein. AlthoughTAA and methionine, lysine and leucine of feeds had significant influence on uAA,uMethionine, uLysine and uLeucine contents, in accordance with the conclusions of Lebzien (1997), it was impossible to calculate the AA pattern of uAA from the AA patternof feeds as the degradation and synthesis of AA in rumen fermentation and in in vitroincubation are influenced by so many factors.

The results imply that the in vitro incubation conditions in the present experiments arequite similar to the average rumen fermentation with respect to the amino acid composition

of duodenal protein and the rumen microbial protein, indicating that the in vitroincubation technique could be a valid method for the estimation of uAA, utilizableMethionine, uLysine and uLeucine of feeds for ruminants.

Comparison of amino acid composition of incubation residues from Expts. 1 and 2

For 27 identical feeds uAA- and uCP-content were determined using as well rumen fluidfrom a cow as rumen fluid from a sheep. Statistical analysis indicates that there was asignificant regression relationship between uAA content of these feeds determined withrumen fluid of a sheep and that with rumen fluid of a cow (Fig. 3).

There was also a significant regression relationship between uCP of feeds determinedwith rumen fluid of a sheep and that with rumen fluid of a cow (Fig. 4). This was also valid

for the estimated amounts of uMethionine, uLysine or uLeucine per kg incubated feed DMdetermined with rumen fluid of a sheep and those with rumen fluid of a cow respectively(uMethionine: r 2 ¼ 0:59; p


10/11

correlated, but it has to be stated that the contents of uAA and uCP of the feeds determinedwith the rumen fluid of the hay-fed cow were significantly higher than those determinedwith rumen fluid of the sheep fed with the mixed ration (p


11/11

were quite similar to rumen fermentation with respect to mean amino acid compositionof duodenal protein and of rumen microbial protein. These results indicate that thein vitro incubation technique of Zhao and Lebzien (2000) can principally be used forestimating uAA, utilizable Methionine, uLysine and uLeucine of feeds for ruminants. Itshould be proved if larger differences between the regressive calculated and the in vitrodetermined values for some feedstuffs are caused mainly by the inherent error of theuCP-regression as independent reference or mainly by the error of the in vitro method.The TAA and methionine, lysine and leucine of feeds had a significant but not decisiveinfluence on the uAA, uMethionine, uLysine and uLeucine contents and it was notpossible to calculate the AA pattern of incubation residues from the AA pattern of feedsas the degradation and synthesis of AA in rumen fermentation or in in vitro incubationare influenced by many factors. As a rumen-fistulated dairy cow is more expensive than arumen-fistulated sheep and most laboratories do not have fistulated cows, it is suggestedto use a rumen-fistulated sheep fed with a mixed ration as prescribed for the Hohenheimgas-test as the donor of rumen fluid for the estimation of uAA of feeds with in vitroincubation, but that in this case 24 h of incubation seemed to be too long and shorter

incubation periods should be tested. Further standardization of the method should bedone.

References

Clark, J. H.; Klusmeyer, T. H.; Cameron, M. R., 1992: Microbial protein synthesis and flows of nitrogen fractions to the duodenum of dairy cows. J. Dairy Sci. 75, 2304–2323.

GfE (Gesellschaft fu ¨ r Erna ¨hrungsphysiologie), 1997: Zum Proteinbedarf von Milchku ¨ hen undAufzuchtrindern. Proc. Soc. Nutr. Physiol. 6 , 217–236.

Lebzien, P., 1997: Zum Einfluss des Futterproteins auf das Aminosa ¨urenmuster des Proteins amDuodenum von Wiederka ¨uern. U ¨ bers. Tiererna ¨hrg. 25, 137–153.

Lebzien, P.; Voigt, J.; Gabel, M.; Gädeken, D., 1996: Zur Scha ¨tzung der Menge an nutzbaremRohprotein am Duodenum von Milchku ¨ hen. J. Anim. Physiol. a. Anim. Nutr. 76 , 218–223.

Naumann, C.; Bassler, R., 2001. Die chemische Untersuchung von Futtermitteln. Band III,Methodenbuch VDLUFA, VDLUFA, Darmstadt, im Druck.

Tilley, J. M. A.; Terry, R. A., 1963: A two-stage technique for the in vitro digestion of forage crops. J. Brit. Grassl. Soc. 40, 139–155.

Zhao, G.Y.; Lebzien, P., 2000: Development of an in vitro incubation technique for the estimation of the utilizable crude protein (uCP) in feeds for cattle. Arch. Anim. Nutr. 53, 293–302.

Author’s address: Dr P. Lebzien, Institute of Animal Nutrition, Federal Agricultural Research CentreBraunschweig, Bundesallee 50, D-38116 Braunschweig, Germany. Tel.: 0531 5963140; Fax: 0531 596 3199; E-mail: [email protected]


The estimation of utilizable amino acids (uAA) of feeds for ruminants using an in vitro incubation...

Documents

Transcript of The estimation of utilizable amino acids (uAA) of feeds for ruminants using an in vitro incubation...