158 M
. Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168
where, AA 5relevant amino acid; DOM5digestible
digestibility of nutrients was calculated from differ-
x
organic matter; DMI 5dry matter intake; NI5feed-N
ences in nutrient flow in digesta at the various sites intake exclusive urea-N; a
5AA2nitrogen fraction of the gastrointestinal tract. Estimation of the amino
of the non-urea-N in the duodenal chyme 50.73;
acids was performed according to the procedure of b
5N from relevant amino acid as a percentage of Spackmann et al. 1958, as modified by Weidner
the total AAN and c 5N content of the individual
and Eggum 1966, using an automated amino acid amino acids .
analyzer Beckmann System 6300. 2.4. Experimental groups
2.5. Statistical methods The nine animals served as a trial group, from
The effects of different amounts of the respective which, every week, two animals were allocated to
amino acids on the N-balances allowed an estimation the balance trials for five days. Selection of the
of the actual amino acid requirements of the animals. animals was based on their state of health, feed
This was undertaken, on the basis of the data intake and performance. During this time, leucine or
derived, for leucine and methionine. methionine were withhold alternatively from the
A three-dimensional surface graph was chosen for infused solution, and faeces, urine and milk were
presentation of the data, with the x-axis representing collected separately to establish N-balances. The
the amount of nitrogen in the milk, the y-axis giving cows were milked twice daily. The urine was
the amount of the relevant amino acid at the delivered into 40-l bins containing 250 ml of hydro-
duodenum and the z-axis showing the N-balance chloric acid. Faeces and urine were weighed and
resulting from the combination of x- and y-values. In subsampled daily for analysis. Composite samples of
this way, the results of various infusion trials dealing the feed and left-over were freeze-dried, ground in a
with the same amino acid were combined. Spline 1-mm screen ultracentrifuge-mill, and analyzed for
interpolation of the derived data was carried out to organic matter, crude protein, crude fibre, ether
interpolate a function for the two variables, N in the extract and ash Naumann and Bassler, 1976. Urine
milk and the amount of amino acids at the and milk samples were analyzed for nitrogen using
duodenum, onto a rectangular grid by using the SAS the Kjeldahl-method Naumann and Bassler, 1976.
G3GRID procedure SAS GRAPH User’s Guide, Milk weights were recorded daily at each milking.
1988. Milk samples of morning and afternoon milkings
were taken on day two and day five of the infusion period, preserved with potassium dichromate and
3. Results and discussion
stored at 4 8C until analyzed for lactose, protein and
fat Infrared analysis Foss Electric, Milko Scan 104 Digestibility values for the crude nutrients of the
A B. solid food are given in Table 2. The infusions had no
Following the 11 infusion periods, the flows of significant P
0.05 effect on the digestibility of the amino acids at the duodenum resulting from rations 1
crude nutrients. Dry matter content of the faeces and 2 were estimated by duodenal digesta collection.
varied between 13 and 16 during the infusions. At A 20-g amount of chromic oxide, mixed with wheat
infusion rates of more than 2000 g hydrolysed flour, was placed in the rumen four times a day.
sucrose per day, the dry matter content of faeces fell Marker administration started nine days prior to the
to 11. No increase in the sugar content of the sampling period. Duodenal samples were collected
faeces was detected. High amounts of monosac- every 2 h for five consecutive days. The samples
charides in the large intestine might have increased were stored at
2188C and used for subsequent microbial protein synthesis, in which case, the
analyses. Concentration of chromium in faecal and digestibility of crude protein might have been ex-
duodenal samples was determined by atomic absorp- pected to decrease. However, this was not the case in
tion spectroscopy Perkin-Elmer Model 400 accord- these experiments. The trials were based on the
ing to the method of Williams et al. 1962. The assumption that an insufficient supply with a single
M . Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168
159 Table 2
Digestibility of the crude nutrients in the rations n 54
Ration Organic
Crude Ether
Crude N-free
matter protein
extract fibre
extract 1
74.5 62.6
65.4 63.2
62.5 66.6
76.7 62.1
77.2 64.0
2 74.3
61.8 66.1
62.8 63.2
66.9 76.6
62.3 76.8
61.5
essential amino acid leads to a negative effect on the acid flow calculated according to Rohr and Lebzien
N-balance, independent of the total N-supply. N- 1991 were less than 10. Nevertheless, for some
balances and productive N in relation during the amino acids, there is considerable imprecision. This
various trial periods are given in Table 3. The results in problems in estimating the duodenal flow
measured amounts of amino acids at the duodenum for single amino acids precisely. Based on the
from rations 1 and 2 are given in Table 4. To duodenal flow of amino acids measured at the
determine if it is possible to obtain an accurate duodenum and the estimated requirement at the
calculation of the flow of individual amino acids at duodenum for individual amino acids, the rank of
the duodenum, based on the characteristics of the limiting amino acids was given as leucine, iso-
rations, the measured amino acid flow at the leucine, methionine, valine, histidine, threonine,
duodenum was compared with calculations based on lysine, phenylalanine and arginine. These findings
equations by Rohr and Lebzien 1991 and Hvel- are in contrast to those of other authors, where, in
plund and Madsen 1989. It can be seen in Table 4 general, methionine and lysine were considered as
that the differences between the measured total first resp. second-limiting amino acids Schwab et
amino acid flow at the intestine and the total amino al., 1976; Clark et al., 1977; Rogers and McLeay,
Table 3 N-balances and productive N of all animals during the trial periods g five days
Period Animal
AA-deficiency N-intake
N-excretion N-balance
Productive N N-balance 1milk-N
1 1
Leu 1900.1
1696.0 204.1
698.7 2
Leu 1900.1
1842.0 58.1
586.6 2
3 Met
1904.1 1818.7
85.5 607.0
4 Met
1904.1 1892.7
11.4 573.6
3 3
Met 1752.5
1751.2 1.3
543.1 5
Leu 1979.5
1891.3 88.1
701.4 4
1 Met
1734.5 1778.6
244.4 504.5
4 Met
1739.6 1724.3
15.3 546.6
5 3
Leu 1764.6
1768.3 23.8
523.5 5
Leu 1910.3
1856.8 53.5
660.2 6
1 Met
1745.2 1771.1
22.9 535.7
2 Met
1719.5 1777.1
257.6 491.0
7 6
Leu 2069.1
1946.9 122.2
722.6 9
Leu 1921.9
1902.8 19.1
646.0 8
1 Met
1774.1 1804.5
230.4 502.2
3 Leu
1816.3 1831.7
215.4 514.7
9 5
Leu 1943.5
1803.0 140.4
713.3 6
Leu 1940.9
2064.4 2121.4
442.4 10
5 Leu
1804.2 1895.9
291.7 507.3
6 Met
1907.0 1863.7
43.3 566.3
11 7
Leu 1955.9
1839.6 116.3
624.9 8
Leu 1936.4
1827.9 108.5
692.6
160 M
. Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168 Table 4
Measured versus estimated flow of amino acids at the duodenum [according to Rohr and Lebzien 1991 and Hvelplund and Madsen 1989, respectively]
Measured AA-flow, calculated according to:
g day Rohr and Lebzien
Hvelplund and Madsen C Hvelplund and Madsen R
g day Difference
g day Difference
g day Difference
Ration 1
Arginine 97.5
82.5 15.0
77.6 19.9
110.9 213.4
Histidine 40.1
40.4 20.3
26.4 13.7
41.1 21.0
Isoleucine 95.2
89.5 5.7
86.6 8.6
111.1 215.9
Leucine 152.1
156.3 24.2
124.1 28.0
155.9 23.8
Lysine 132.5
124.6 7.9
102.1 30.4
142.6 210.1
Methionine 33.2
38.5 25.3
27.7 5.5
39.2 26.0
Phenylalanine 108.6
89.5 19.1
76.8 31.8
93.8 14.8
Threonine 99.9
91.2 8.7
87.9 12.0
107.2 27.3
Valine 108.2
98.3 9.9
99.6 8.6
127.6 219.4
Alanine 122.1
119.4 2.7
110.8 11.3
138.2 216.1
Aspartic acid 204.4
186.0 18.4
111.8 92.6
231.5 227.1
Cysteine 33.3
22.6 10.7
19.9 13.4
24.9 8.4
Glutamic acid 257.5
249.3 8.2
209.4 48.1
299.9 242.4
Glycine 193.2
117.7 75.5
91.5 101.7
134.3 58.9
Proline 80.9
87.8 26.9
62.7 18.2
86.7 25.8
Serine 93.4
84.3 9.1
86.0 7.4
108.3 214.9
Tyrosine 90.1
77.1 13.0
76.4 13.7
101.5 211.4
o 1942.2
1755.0 187.2
1477.3 464.9
2054.7 2112.5
Ration 2
Arginine 81.7
76.0 5.7
71.3 10.4
102.0 220.3
Histidine 33.6
37.2 23.6
24.3 9.3
37.8 24.2
Isoleucine 82.2
82.4 20.2
79.7 2.5
102.2 220.0
Leucine 129.8
143.9 214.1
114.1 15.7
143.4 213.6
Lysine 112.8
114.7 21.9
93.9 18.9
131.2 218.4
Methionine 29.1
35.5 26.4
25.4 3.7
36.1 27.0
Phenylalanine 95.2
82.4 12.8
70.6 24.6
86.2 9.0
Threonine 85.7
84.0 1.7
80.8 4.9
98.6 212.9
Valine 88.2
90.5 22.3
91.6 23.4
117.4 229.2
Alanine 104.9
110.0 25.1
101.9 3.0
127.1 222.2
Aspartic acid 175.8
171.2 4.6
102.9 72.9
212.9 237.1
Cysteine 28.4
20.8 7.6
18.3 10.1
22.9 5.5
Glutamic acid 223.5
229.5 26.0
192.6 30.9
275.8 252.3
Glycine 173.4
108.4 65.0
84.2 89.2
123.5 49.9
Proline 69.4
80.8 211.4
57.7 11.7
79.7 210.3
Serine 80.7
77.6 3.1
79.1 1.6
99.6 218.9
Tyrosine 78.6
71.0 7.6
70.3 8.3
93.3 214.7
o 1673.0
1615.9 57.1
1358.7 314.3
1889.7 2216.7
R 5‘‘roughage-diet’’, C5‘‘concentrate-diet’’, as mentioned by Hvelplund and Madsen 1989.
1977; Rogers et al., 1979; Ayoade et al., 1982; importance of leucine and methionine as first-limit-
Casper and Schingoethe, 1986; Rulquin, 1987; Geip- ing amino acids. It has to be taken into account that
ing and Menke, 1989; Geiping and Menke, 1991; differences in the extent of supplementation are often
Fraser et al., 1991; Rulquin, 1992. Brandt et al. quite small and, thus, changes in the ranking may
1987, on the other hand, stressed the special occur easily. Due to this, Schwab et al. 1976
M . Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168
161 Table 5
Relation of amino acid supply at the duodenum to the estimated amino acid requirement Rohr and Lebzien, 1991 at the duodenum of the lactating dairy cows in
Period Animal
ARG HIS
ILE LEU
LYS MET
PHE THR
VAL 1
1 198.7
138.0 126.3
120.1 153.4
130.9 161.3
140.7 135.7
2 191.5
132.4 120.2
114.7 146.2
124.4 153.9
135.4 129.7
2 3
191.8 124.4
120.3 108.4
144.7 102.3
154.1 135.4
122.8 4
185.6 119.6
114.8 103.8
138.2 97.5
146.7 130.1
117.7 3
3 158.5
87.7 103.8
97.0 121.5
107.7 131.9
112.9 105.1
5 189.2
129.3 115.4
110.7 140.9
119.0 143.7
121.5 124.5
4 1
159.3 112.6
104.3 97.7
122.1 87.9
130.1 112.2
105.7 4
161.0 113.2
104.8 98.4
123.3 89.2
135.5 116.6
106.5 5
3 161.9
113.8 106.5
98.9 124.0
110.0 136.3
117.2 107.1
5 173.5
130.3 116.4
111.7 128.5
120.1 143.3
123.9 125.6
6 1
163.8 119.1
110.9 103.7
129.7 90.8
138.8 118.9
112.0 2
159.5 106.5
105.1 92.7
119.4 88.7
134.1 116.0
100.7 7
6 176.9
135.9 120.9
116.0 146.8
124.9 136.2
113.6 130.2
9 173.0
127.5 113.5
109.0 131.0
117.1 138.3
116.4 122.7
8 1
159.8 108.7
107.7 94.5
119.1 88.2
132.6 114.2
102.5 3
158.7 113.6
106.1 99.1
119.5 109.8
127.9 109.3
107.1 9
5 183.1
128.2 115.7
110.4 133.0
119.3 145.4
129.2 124.8
6 184.3
136.5 123.2
117.8 134.5
127.3 146.8
127.5 132.4
10 5
149.3 107.5
98.3 92.7
108.6 101.9
123.8 108.0
100.3 6
192.6 133.2
121.7 115.1
147.3 102.1
155.1 136.2
130.7 11
7 195.3
141.1 129.0
122.6 145.0
133.2 158.0
138.4 138.0
8 181.4
123.4 111.5
106.3 131.4
114.8 138.1
120.9 106.3
Fig. 1. Dose–effect area interpolated for methionine.
162 M
. Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168
defined several amino acids as being co-limiting for methionine or leucine in milk and body tissue at
milk production. different levels of methionine leucine inclusion. As
long as the amino acid requirement for milk protein 3.1. N balance in correlation with the amount of
synthesis is not met, body protein is metabolised and leucine and methionine at the duodenum and the
N-balance is negative. If the amount of amino acid at amount of N in milk
the duodenum is higher than that required for milk protein synthesis, if no other nutrient is limiting, and
The amino acid supply at the duodenum as a result if there is still a capacity for body protein synthesis,
of both rations and infusions was calculated and the then surplus will be stored as body tissue protein and
results are given in Table 5 as a percentage of the the N balance will increase. Amino acids that cannot
calculated requirement Rohr and Lebzien, 1991 at be used for protein synthesis will increase the level
the duodenum. The relations between the three of N excretion in the faeces and or in the urine.
parameters N balance, amount of amino acids at the Therefore, requirements for milk protein synthesis,
duodenum and N in the milk are presented graphical- including the animal’s maintenance requirements,
ly. These ‘‘dose–effect areas’’ for leucine and should be defined at zero N balance. Rulquin et al.
methionine at various milk yields are shown in Figs. 1993 postulated that amino acid requirements are
1 and 2. A three-dimensional presentation was met ‘‘when milk protein responses are slightly below
chosen to include all three parameters in one graph. the maximum attainable values’’.
Based on the results of the infusion trials, by The contour lines of the dose–effect areas, the
interpolation, value-triplets for all amino acid so-called ‘‘isoboles’’, are defined as the number of
amount N balance N in the milk combinations combinations with similar effects. Of special interest
were calculated. The figures show a marked differ- are isoboles where N-balances are zero ‘‘zero-iso-
ence in the proportionate partition of an increment of bole’’, because these curves describe, as explained
Fig. 2. Dose–effect area interpolated for leucine.
M . Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168
163
before, the actual amino acid requirements of the and Ørskov 1982 calculated a utilisation level of
animal at the duodenum for a certain milk protein 80 for absorbed microbial amino acids. MacRae
yield Figs. 3 and 4. and Beever 1997 emphasised the relation of amino
The points in Figs. 3 and 4 represent the amounts acid requirement to amino acid supply for its utilisa-
of methionine leucine at the duodenum, calculated tion.
from the relations shown in Figs. 1 and 2, giving a zero N balance at milk protein yields ranging from
3.2. N-balance in correlation with the percentage 618 to 764 g per day. Higher or lower levels of
of amino-acid supply amino acids at the duodenum per g of milk protein
would result in positive or negative N-balances and The trials were based on the assumption that
not in more milk protein, and would influence the supplying the animals with an amino acid amount of
calculated efficiency of conversion of absorbed 100 of the estimated requirement would result in a
amino acids into milk protein Rulquin et al., 1993; zero N-balance if the estimated requirement is
MacRae and Beever, 1997. correct. N-balances in relation to the amino acid
Under the supposition that the concentrations of supply as a percentage of the estimated requirement
methionine and leucine in milk protein are 2.5 and for methionine and leucine are given in Figs. 5 and
9.6, respectively, and that the absorption rates of 6.
duodenal methionine and leucine are 80 and 87 From a statistical point of view, linear functions fit
Lebzien and Rohr, 1994, efficiencies of conversion nearly as well through the points as the chosen
of absorbed amino acids into milk of 78 and 86 functions. The reason for using these sophisticated
can be calculated. These values are quite high. Storm curves is that, in adult cows with limited milk
Fig. 3. Estimated amounts of methionine at the duodenum at different milk-N yields required for zero N balance.
164 M
. Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168
Fig. 4. Estimated amounts of leucine at the duodenum at different milk-N yields required for zero N balance.
production, N-balance cannot increase endlessly with vidual amino acids can therefore be calculated using
increasing amino acid supply. There is, however, a split-requirement values for maintenance and milk
limitation to this approach, namely, the method of yield, and the amino acid patterns of body protein,
interpolation is likely to introduce an error of intestinal chyme and milk protein Rohr and Leb-
estimation, resulting from curvilinearity of the re- zien, 1991.
sponse, particularly when extrapolating beyond the extremas of the range of data.
3.3. Estimations of leucine- and methionine It can be seen that, in the case of methionine, the
requirements for lactating dairy cows estimated requirement for a zero N-balance was too
high. Feeding the animals with 100 of the esti- In Table 6, methionine requirements, estimated by
mated requirement Rohr and Lebzien, 1991 for dose–effect curves Fig. 3 and by N-balances Fig.
methionine resulted in a positive N-balance, imply- 5, are compared with factorial calculations of
ing that the methionine requirement was overesti- methionine requirements.
mated by approximately 10. Whether this overesti- Estimated values for intestinal absorption of
mation is a result of 1 underestimating the intesti- methionine in the literature vary over a broad range,
nal absorption. 2 underestimating the intermediary from merely 65 Hagena, 1985 to 90 Burroughs
utilisation or 3 a combination of the two parame- et al., 1975, with values for intermediary utilisation
ters intestinal absorption and intermediary utilisation varying between 60 ARC, 1984 and 95 Bur-
needs further investigation. roughs et al., 1975. Storm et al. 1983, Van
In the case of leucine, the calculated requirement Bruchem et al. 1989 and Lebzien and Rohr 1994
agreed well with the estimated requirement. measured an absorption of 87–90. As seen in
The above-mentioned requirements of the indi- Table 6, an intestinal absorption of 90 seems to be
M . Iburg, P. Lebzien Livestock Production Science 62 2000 155 –168
165
Fig. 5. N-balances in relation to methionine supplementation in of the estimated requirements dairy cows.
more appropriate, as using this value produced the 1975. Literature data given by Rulquin et al.
values derived using the N-balance method. How- 1993 varied between 31 and 52 g of methionine per
ever, an absorption of 80 for the factorial calcula- day for a cow with a BW of 600 kg and that
tion led to values that closely resembled the esti- produced 30–35 kg of milk.
mates of Rulquin et al. 1993. Rulquin et al. 1993 For leucine, an assumption of an intestinal absorp-
stressed that, in the factorial approach, the choice of tion rate of 87 Lebzien and Rohr, 1994 and an
amino-acid composition of product, the absorption intermediary utilisation for milk production of 80
rate and the efficiency of utilisation largely de- seems to be appropriate Table 7. In the literature,
termines the final requirement values. estimated values for the intermediary utilisation vary
Burroughs et
al. 1975
reported a
total between 60 ARC, 1984 and 80 GfE, 1986,
methionine requirement of 30.8 g for a dairy cow values for absorption are 87 and 88 Storm et al.,
with a BW of 550 kg and an average milk yield of 1983; Hvelplund and Hesselholt, 1987; Van Bruchem
30.0 kg day in that publication, calculations were et al., 1989; Lebzien and Rohr, 1994.
based on an average content of 3.2 milk protein. No endogenous N-losses were considered in that
¨ calculation, in contrast to Gunther 1987, who
4. Conclusion
