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Animal Science Journal

(2003) 74, 457469



457

Blackwell Science, LtdOxford, UKASJAnimal Science Journal1344-39412003 Blackwell Publishing Asia Pty LtdDecember 2003746457469Review Article

Amino acid nutritionT. ISHIBASHI and C. YONEMOCHI

Correspondence: Teru Ishibashi, Japan Scientic Feed Asso-
ciation, 2-6-16 Sinkawa, Chuo-ku, Tokyo 104-0033, Japan.
(Email: teru-ishibashi@kashikyo.or.jp)
Received 17 March 2003; accepted for publication 29 July
2003.

REVI EW ARTI CLE

Amino acid nutrition in egg production industry

Teru ISHIBASHI and Chisato YONEMOCHI

Japan Scientic Feeds Association, Chuo-ku, Tokyo, Japan

ABSTRACT

The egg production industry is facing various problems that need to be solved. For amino acid nutrition to achieve scientic
and economical feeding of laying hens, it is necessary to elucidate the content, digestibility, or availability of nutrients of
feedstuffs and feeds and the requirement of amino acids for laying hens. In addition, improvement to quality of eggs and
meat of spent hens, methods of management and development of new feedstuffs are essential. For sustainable animal
production, decrease in excreta and animal welfare should be studied. The real-time determination of content and digest-
ibility of amino acids in feedstuffs are essential for formation of feeds. Recent advances in the near infrared reectance
analysis will be able to determine the content, digestibility and availability of nutrients in feedstuffs and feeds, if we have
a supplemental amount of conventional analysis to dene the calibration population. The amino acid requirements are
affected by various factors. Therefore the method to quickly and exactly determine amino acid requirements in response
to various factors is necessary. By using plasma free amino acid concentration as a criterion, it is possible to determine
amino acid requirements in various conditions of laying hens within a short experimental period, repeatedly using the same
animals. Because the amino acid requirements differ among individual animals, it should be expressed as grams per hen
per day. Practically, it is impossible to formulate various feeds for individual hens. The various expressions have been devel-
oped and these expressions have advantages and disadvantages. The nitrogen excretion of laying hens is easily reduced
by reducing dietary nitrogen levels and restricting the feed intake. The availability of amino acid may be improved by feed-
ing management, and supplementing enzyme, but the quality of eggs and meat of spent hens and welfare of laying hens
are not affected by amino acid nutrition.

KEYWORDS:

amino acid requirements, laying hens, nitrogen excretion, plasma amino acid concentration, qual-

ity of eggs.

INTRODUCTION

The total number of laying hens has increased 3.2%
per year since 1995 and the amount of eggs reached
51.11 million tons, which included 3.74 million tons
(6.4% of total) of eggs for hatchery, in 2000. Egg con-
sumption per head is increasing worldwide except for
the USA, Canada and Australia (USDA 2001; Fig. 1).
In these countries, egg consumption decreased after
the 1970s because the cholesterol in egg yolk is
thought to cause heart and circulatory disease. The
Japanese consume the most eggs per head per day in
the World. Since 1995, an average 328 eggs per head
are consumed annually, which shares approximately
10% of the total protein intake of the Japanese
(Table 1). The amount of feed consumed by laying
hens is 6 million tons annually. More than 80% of
feedstuffs for laying hens are imported from abroad,
which is one of the largest factors affecting negative
balance of the trade. Some of the feedstuffs are com-
peting with human foods. Therefore, it is most impor-
tant to study the effective use of feedstuffs not only for
the improvement of negative balance of trade in
Japan, but also the decrease in competition between

T. ISHIBASHI and C. YONEMOCHI

Animal Science Journal

(2003) 74, 457469

458

feeds and foods in the world. In order to rear livestock
efciently and economically, the information on con-
tents, digestibility or availability of nutrients in feed-
stuffs and feeds, and the exact requirements for
nutrients to livestock, is essential. In addition to the
improvement of effective use of feedstuffs, the egg
production industry is facing a number of difcult
problems, which require solving, such as decrease in
excretion of nitrogen and phosphorus for the sustain-
able animal industry, supply of safe and tasty eggs and
meats, development of new feedstuffs, technology of
feeding management and animal welfare.

DETERMINATION OF CONTENTS
OF AMINO ACIDS IN FEEDSTUFFS
AND FEEDS

The average content of nutrients in feedstuffs is avail-
able from Standard Tables of Compositions of Feed-
stuffs in Japan (National Agricultural Research
Organization 2001). However, the nutritive values of
feedstuffs are not constant, and differ depending on
factors such as the area of origin, harvest period, man-
agement and process after harvest. In addition to
these factors, the nutrient content in feedstuffs varies
among different batches of the same feedstuffs. Using
conventional chemical methods, it is impossible to
determine the nutrient content of feedstuffs on a real-
time basis. Therefore, there is a need for a new real-
time analysis of the formation of feeds. Near infrared
reectance analysis (NIRA) is not a new method, but
recent NIRA may be the answer for providing rapid
and accurate analysis of not only the nutrient content,
but also the digestibility of energy and amino acids
(Van Kempen & Simmins 1997; Dudley-Cash 1998).
It is an advantage of NIRA that it can very rapidly and
accurately determine the nutrient content of feed-
stuffs that are within the calculation population.
However, NIRA has the disadvantage that it requires a
suboptimal amount of conventional

in vivo

and/or

in
vitro

analysis in order to adequately dene the calibra-
tion population.

DETERMINATION OF DIGESTIBILITY
AND AVAILABILITY OF NUTRIENTS
IN FEEDSTUFFS AND FEEDS

All nutrients in feeds are not always used efciently.
Some are excreted into feces without being digested
and absorbed, and other absorbed nutrients are
excreted into urine without being metabolized in the
body. Therefore, it is important to determine whether
the ingested nutrients are digested, absorbed, and
metabolized, when the nutrients are slightly in excess
of requirements.

Digestibility of nutrients in feedstuffs
and feeds

For the economical and scientic formulation of
poultry diets, the data of contents and digestibility or
availability of nutrients in feedstuffs are essential.
Because poultry excrete feces and urine together
from the cloaca, it is necessary to separate feces from
urine by setting a cannula to determine the digestibil-
ity of feeds. By the development of the reversed rec-
tum articial anus (Isshiki & Nakahiro 1988), it is
easy to maintain a large number of operated hens
which continue high egg production rate without
special daily care for a long period. The digestibility of
crude protein (CP, N

6.25

), determined from the
difference between CP in diet and feces, are ap-
parent digestibility and not true digestibility of CP. In
order to determine the true digestibility of CP, the

Fig. 1

Eggs produced per layer after 1925 (USDA 2001).
250
200
150
100
1925 1950 1975 2000
Year
E
g
g

p
r
o
d
u
c
e

(
/
l
a
y
e
r
)

Table 1

Annual production and consumption of eggs in 2000
(USDA 2001)

Country Production
(milion tons)
Consumption
(eggs/head)
Total 51.11


China
USA 258
Japan 328
France 264
Germany 223
Italy 219


Data unavailable for these sections.

Amino acid nutrition

Animal Science Journal

(2003) 74, 457469

459

endogenous CP derived from decomposed tissues and
intestinal microora has to be subtracted from that in
feces. To determine endogenous nitrogen, we can use
three kinds of collected feces: (i) after feeding a diet
without CP and amino acids, (ii) after feeding a diet
with graded levels of CP, and (iii) with the removal of
a diet. In the case of feeding the diet with graded lev-
els of CP, the excretion of nitrogen at zero point of
dietary CP is estimated by extrapolation method. This
method is the least stressful to hens.

Availability of dietary amino acids
in feedstuffs and feeds

Some amino acids are excreted into urine when
excess amino acids are fed to chickens. The urinary
amino acids must be corrected to evaluate a true
availability of amino acids. Yamazaki and Kaku
(1988) developed the new method to overcome this,
which was modied to determine true metabolizable
energy (ME) in feedstuffs (Sibbald 1979). This
method does not need hens with an articial anus.
The endogenous amino acids are obtained by deter-
mining amino acids in the excreta from chickens
fasted or fed a nitrogen-free diet for more than 48 h.
The values obtained using this method coincide with
those determined using roosters with an articial
anus. Although the digestibility of feedstuffs fed as a
sole source tends to be lower than that obtained by
feeding a mixture of feedstuffs, the digestibility of
mixed feedstuffs is calculated by adding each value
of feedstuffs, which is not affected by sex and age of
poultry. The digestibility of crystalline amino acids is
estimated to be 100% (Yamazaki & Kaku 1988).

Digestibility of nutrients of feedstuffs
and feeds at the end of the ileum

In pigs, there are some differences between the digest-
ibility determined using feces and digesta at the end of
the ileum (Furuya

et al

. 1979). In poultry, there were
differences between the digestibility of amino acids
determined using excreta and digesta at the end of the
ileum. The degree of differences varied among feed-
stuffs and individual amino acids (Ravindran & Boy-
den 1999). To determine the digestibility at the end of
ileum, samples have to be collected from the hens t-
ted with a cannula at the end of ileum or digesta at the
end of the ileum after being killed. The volume of
cecum, colon and rectum of poultry is small compared
to other herbivores, the role of intestinal microora in
the digestion of nutrients at the lower part of digestive
lumen is small.

Factors affecting digestibility of nutrients
of feedstuffs and feeds

The digestibility of nutrients in feedstuffs is affected by
various factors, such as process of feedstuffs and coex-
isted components, tannic acid (Yamazaki & Kaku
1988) and enzymes contained in feedstuffs (Acamovic
2001).
The suggestion by Dintzis

et al

. (1998) has to be con-
sidered to determine the exact digestibility of CP. The
digestibility of CP differed between CP calculated from
total nitrogen determined by the Kjeldahl method and
recovered amino acids plus ammonium nitrogen
determined by amino acid analysis. These differences
might be smaller in the digesta in the ileum than in
feces because the amount of ammonium nitrogen in
the digesta in the ileum might be smaller than in feces.

Determination of digestibility
of feedstuffs

in vitro

The determination of digestibility of nutrients in feed-
stuffs

in vivo

is time consuming and laborious, and the
digestibility of only one sample is determined from
one trial. Therefore, the rapid and accurate analysis
method, by which the digestibility of many samples is
estimated simultaneously within a short period, is pre-
ferred. The

in vitro

method has been developed to
determine the digestibility of dry matter, digestive
energy and CP using the liquid from the small intes-
tine of a pigs attached intestinal cannula (Furuya

et al

.
1979). The obtained values coincide with those at the
end of ileum. This method using the intestinal uid of
pigs is applicable to laying hens (Sakamoto

et al

. 1980).
However, to increase the accuracy of this method,
some standard samples, of which

in vivo

digestibility is
known, should be included in each

in vitro

experiment
and their values should be used for corrections. For
the effective use of NIRA for real-time estimation of
the digestibility of feedstuffs in the feed-formulating
industry, numerous data on the digestibility of nutri-
ents of feedstuffs determined by this method may be
available, and is expected to be accumulated further.

AMINO ACID REQUIREMENTS
OF LAYING HENS

Approximately 40 years ago, poultry diets were for-
mulated largely on CP. As observed by Osborne and
Mendel (1914), the nutritive values of CP differ
among their sources. For example, when three corn-
based diets with whole egg powder, sh meal or

T. ISHIBASHI and C. YONEMOCHI

Animal Science Journal

(2003) 74, 457469

460

soybean meal as main sources of CP were supplied, the
maximum performance of broiler chicks was achieved
at 15.0, 16.5 and 20.4% of CP, respectively. By adding
limiting amino acids to the soybean meal diet, chicks
achieved the same performance as those fed the whole
egg powder diet (Koide

et al

. 1992). These results indi-
cate that the scientic protein nutrition of poultry is
no longer based on the CP content of the diet. The
dietary levels and biological availability of essential
amino acids have to be considered, together with suf-
cient dietary levels of nonessential amino acid nitro-
gen at the cellular and molecular levels, and with all
elements needed to synthesize all body and egg pro-
teins efciently and economically.
Historically, Rose and Meyer (1936) conrmed that
animals could be reared on a diet with amino acid as
the only nitrogen source. First, approximately 60% of
egg production rate, which was commercially average
egg production at that time, was achieved by feeding a
diet containing crystalline amino acids as a sole nitro-
gen source (Fisher & Johnson 1956). The egg produc-
tion rate of recent commercial hens exceeds 90% at
the peak egg production stage as shown in Fig. 2. This
high egg production rate was maintained for more
than six months on the amino acid diet (Ishibashi &
Kametaka 1984).
Numerous studies have been conducted to deter-
mine exact amino acid requirements and the results
are evident in feeding standards. The requirements of
CP and amino acids in the feeding standards are sum-
marized in Table 2. The recommended feed intake
decreased from 110 to 100 g in version 9 by the
National Research Council (NRC 1994), and the
amino acid requirements expressed as percentages of
diet increased. The amino acid expressed as grams per
hen decreased with advancing versions. The egg pro-
duction rate increased after the 1920s and the number
of eggs per hen is increasing at the rate of 1.25 eggs
annually over the last ve years (Table 2). In spite of
the increment of egg production rate, the amino acid
requirement decreased with advancing versions.
These decreases in amino acid requirements might be
caused by determination of exact requirements of
amino acids.
In the feeding standards, only one pattern of amino
acid requirement is shown. However, the amino acid
requirements of laying hens are affected by various
factors such as genetic, environmental, physical, and
managing factors (Ishibashi 1990). The genetic, envir-
onmental and managing factors are not so large
among present stocks (Pobrow & Morris 1974). The
amino acid requirements are most drastically affected
by egg production rate. The egg production rate is not
constant throughout the laying period and increases to
more than 90% at 17 weeks of age (Fig. 2). After this,
egg production rate and the quality of eggs decreases,
and egg size increases with advancing age. Because
amino acid composition of egg white and egg yolk
does not change, the amino acid requirements
decrease with advancing age and the decrease of egg
production rate. At the peak of egg production rate for
2030 weeks of age, the CP requirement was esti-
mated to be 15.7%. The CP requirements decreased to

Fig. 2

(


), Average bodyweight (g); (


), feed intake
(g/week); and (


), egg production rate (%) cited from
pamphlets of nine breeding companies (average values of
data of seven breeding companies).
B
o
d
y

w
e
i
g
h
t

(
g
)
2000
1500
1000
500
0
20 40 60 80
0
20
40
60
80
100
Age (weeks)
E
g
g

p
r
o
d
u
c
t
i
o
n

(
%
)

Table 2

Protein and amino acid requirements of laying hens
recommended by NRC (% of diet) in 6th to 9th revised edition
(NRC 1971, 1977, 1984, 1994)

1971 1977 1984 1994
Feed intake (g/d)


110 110 100
ME (kcal/kg diet) 2850 2850 2900 2900
CP 15.0 15.0 14.5 15.0
Arg 0.8 0.8 0.68 0.70
Gly

+

Ser 0.5 0.50
His 0.22 0.16 0.17
Ile 0.5 0.5 0.50 0.65
Leu 1.2 1.2 0.73 0.82
Lys 0.5 0.60 0.64 0.69
Met

+

Cys 0.53 0.50 0.55 0.58
Phe

+

Tyr 0.80 0.80 0.83
Thr 0.4 0.4 0.45 0.47
Trp 0.11 0.11 0.14 0.16
Val 0.5 0.55 0.70

NRC, National Research Council; ME, metabolizable energy; CP,
crude protein;


not available.

Amino acid nutrition

Animal Science Journal

(2003) 74, 457469

461

14.1% for 6080 weeks of age and 12.4% after then
with a decreasing egg production rate (Okazaki

et al

.
1994). In these diets, amino acid levels ranged from
130, 115 and 100% of the NRC requirement, respec-
tively. These depressions are mainly caused by
decrease in egg production rate, because the body-
weight gain is less than 200 g and the amino acid
requirements for maintenance do not increase after
20 weeks of age. Therefore, the CP level of diet for lay-
ing hens should be arranged according to the egg pro-
duction rate and other factors. In order to formulate
layer diets, it is very important to determine the exact
requirements of CP and amino acids. In practice, how
to express these requirements is also important.

Determination of amino
acid requirements

Throughout the study of animal nutrition, researchers
have tried to develop improved methods for determin-
ing amino acid requirements including using body-
weight gain, production, nitrogen balance and feed
efciency. These parameters are insensitive for adult
animals, laborious and expensive, and require a long
period and large number of animals to be studied.
There are two methods which are able to predict
amino acid requirements within a short period and are
more sensitive for adult animals. In these methods the
oxidation of indicator amino acid labeled with radio-
active or stable isotope and the response of plasma-
free amino acid concentrations to the changes in
dietary amino acid levels are used as parameters.

Indicator amino acid oxidation method

In the indicator amino acid oxidation method, amino
acids labeled with radioactive or stable isotopes are
used as an indicator of oxidation. In both cases, the
used indicator isotope is the same as or different from
that to be determined as the requirement. When the
same indicator amino acid as the test amino acid is
used, the method is based on the principle that when
intake of the test amino acid is limiting, protein syn-
thesis and oxidation of the test amino acid is low. As
the test amino acid intake exceeds the requirement
levels, the excess test amino acid is oxidized and
expired as CO

2

. The requirements of Lys, Leu, and
Val of broiler chicks were determined in this method
(Ishibashi

et al

. 1977a,b).
In the case where the indicator amino acid is differ-
ent from the test amino acid, the method is based on
the principle that when intake of the test amino acid is
limiting, protein synthesis is low, and all other amino
acids are excess and oxidized. As the test amino acid
intake increases, protein synthesis increases and oxi-
dation of other amino acids decreases. Above the test
amino acid requirement, additional intake of test
amino acid will not increase protein synthesis, and the
oxidation of other amino acids remains constant. By
using a radioactive or stable isotope in humans, the
oxidation of one of the other amino acids (i.e. the indi-
cator) can be measured at a given intake of the test
amino acid. In both cases, the break points change
from constant to increasing oxidation of the same kind
of amino acid and from decreasing to constant indica-
tor amino acid oxidation are deemed the requirement
and are statistically assessed (Chamruspollert

et al

.
2002; Tabiri

et al

. 2002a,b).
The determined value by feeding test is an average
amino acid requirement for a long feeding period.
Within a short period, the amino acid requirement
changes with changing physical, environmental con-
ditions and egg production rate. The indicator amino
acid oxidation method is superior to the feeding test to
determine amino acid requirement, because the
requirement of amino acid is determined at a given
age and egg production stage, 1 or 2 days, compared to
a much longer period for feeding trials, 1 or 2 weeks.
However, radioisotope is expensive, dangerous and
limited to use only in the isotope institute.

Response of plasma concentration of free amino acids
as a criterion

The method using the response of plasma-free amino
acid concentration to the changes in dietary amino
acid levels as an indicator, is able to predict amino acid
requirements within a shorter period than the indica-
tor amino acid oxidation method. The principle of this
method is based on when a given amino acid intake is
less than the requirement, the plasma concentration of
a given amino acid remains low. When the given
amino acid intake exceeds the requirement level, it
increases linearly with increasing intake of the given
amino acid (Fig. 3). The point where the plasma con-
centration of free-given amino acid starts to increase
corresponds with the requirement determined using
the traditional methods in rats (Stockland

et al

. 1970),
chicks (Zimmerman & Scott 1965; Ishibashi 2000),
laying hens (Chi & Speers 1976; Yamamoto & Ishiba-
shi 1996, 1997a,b), pigs (Mitchell

et al

. 1968) and
young men (Young

et al

. 1972).
The plasma concentration of free amino acid
responds quickly, within 2 days, to changes in dietary
amino acid levels and the response is kept for a long

T. ISHIBASHI and C. YONEMOCHI

Animal Science Journal

(2003) 74, 457469

462

period. Therefore, it is possible to predict amino acid
requirement repeatedly using the same animals within
2 days after changing dietary amino acid levels (Yama-
moto & Ishibashi 1997b; Yamamoto

et al

. 1997).
If the amino acids which are not metabolically
related are combined, the requirements of two amino
acids are able to be predicted simultaneously (Ishibashi
2000).
The enzyme activity related to protein synthesis and
degradation in the skeletal muscle of mice responded
within 6 h after changing the dietary CP levels
(Yoshizawa

et al

. 1997). Therefore, the plasma concen-
tration of free amino acids may reect quickly the
response of enzymes in laying hens and other species.
Both methods will alleviate the stress of animals,
shortening the experimental period.

Expression of amino acid requirements

There are ve possible ways of expressing amino acid
requirements. They are expressed as: (1) grams of each
amino acid per animal per day, (2) percentages of diet,
(3) grams of each amino acid per ME, (4) percentage
of dietary CP, and (5) ratios of each amino acid to Lys
requirement.
As shown previously, the amino acid requirement of
laying hens depends on their egg production rate, and
the egg production rate varies among hen. Therefore,
Method (1) is the most precise way to express the
amino acid requirements, but difcult to use in farms
and feed manufacture industry. Method (2) is used
most popularly in practice, but is completely unsatis-
factory because the feed consumption of hens depends
largely on the ME content. In the open-type house
during the summer months, hens fed the same ME
diet consume as little as approximately 20% of feed
per hen per day during winter months. In order to sat-
isfy the amino acid requirement, the percentage of
each amino acid present in the diet should be
increased in summer months.
When the dietary ME is increased and hens con-
sume less feed, and when the dietary ME is increased,
it is deemed to be better to use Method (3) which ties
the amino acid requirements to ME content of the
diet. This method is useful in the practical formulation
of a diet for laying hens.
Recently, Al-Saffar and Rose (2002) indicated that
expressing amino acid supply as a proportion of CP is
preferable in laying-hen nutrition. In Method (4), it is
considered best practice to determine a CP level, in
which, most essential and nonessential amino acids
are provided at any given ME level. This CP level is
adjusted with special supplements such as synthetic
amino acids and protein sources, including sh meal
and soybean meal, in such a way that each essential
amino acid, as closely as possible, meets the require-
ment expressed as a percentage of the dietary CP. This
method is not always right. When nonessential amino
acids, for example 5% of Glu (0.45% as CP), are
increased, the requirements of essential amino acids
were not affected (Ueno 1998).
Baker (1997) explained the ideal amino acid pro-
les in various animal species. In Method (5), the
requirement of each amino acid is expressed as a
ratio to Lys as an ideal amino-acid balance. The Agri-
cultural Research Council (ARC) (1981) discussed
and proposed an ideal amino-acid balance for pigs,
but not for poultry. As mentioned, the amino
acids are used for maintenance, renewal of feathers
and synthesis of egg protein in laying hens. In laying
hens with high egg production rate, the large
amount of dietary amino acids is used for egg protein
synthesis. The amino-acid composition of the egg is
not affected by dietary amino-acid composition and
egg production rate. Therefore, the balance of dietary
amino acid for laying hens may be constant, and it is
deemed to be reasonable to express the requirement
of each amino acid as a ratio to a given amino acid

Fig. 3

Plasma lysine concentration of laying hens, which laid
on the day and the next day of blood sampling, and were fed
diets with 0.45 (


), 0.55 (


), 0.65 (


), 0.75 (


) and 0.85% (


)
lysine at 2 days after changing, successively, dietary lysine
levels.
400
300
200
100
0
500 700 900
Lysine intake (mg/hen/day)
P
l
a
s
m
a

l
y
s
i
n
e

c
o
n
c
e
n
t
r
a
t
i
o
n

(
n
m
o
l
/
m
l
)

Amino acid nutrition

Animal Science Journal

(2003) 74, 457469

463

such as Lys in pigs. In pigs, Lys is most liable to be
limiting and the data of analysis of Lys in feedstuffs
and requirements of Lys are accumulated as shown
in feeding standard for pigs (MAFFRC 1998).
Although Arg is synthesized in pigs, Arg is essential
for poultry. In broilers, Lys requirement is affected by
dietary Arg levels and expressed as a function of
dietary Arg levels and vice versa as follows: (Ueno
1998)
Yl = 0.00565Xa + 0.388

R

2

= 0.996
Ya = 1.55Xl 0.21

R

2

= 0.994
where Yl and Ya are requirements of Lys and Arg
expressed as percentages of diet, and Xa and Xl are
percentages of dietary Arg and Lys, respectively. The
levels of dietary Lys and Arg do not affect the require-
ments of other amino acids. In laying hens, sulfur con-
taining amino acids (Met

+

Cys; SAA) is most liable to
be limiting and Lys is the second-most limiting amino
acid. Therefore, another expression of amino acid bal-
ance should be developed.

Factors affecting amino acid
requirements

The requirements of CP and amino acids are not con-
stant and are affected by many factors (Ishibashi
1990). Among these are management factors; envir-
onmental temperature, housing style (cage or oor
pens), feeding space per hens, feeders, whether or not
the hens are properly debeaked, degree of crowding of
hens in pens, supply of drinking water, disease level in
the ock and physical and genetic factors; size and
breed of hen, and dietary factors; energy content of
diets and relationship among amino acids, such as
antagonism and imbalance.
Provided these factors are controlled satisfactory, the
amino acid requirements of laying hens are mainly
affected by egg mass.
The amino-acid requirement of laying hens is a
sum of amino acids demanded for growth of body
tissues and feathers, maintenance, and production of
an egg. Assuming that 18% of bodyweight gain is
protein, and the efciency of converting the protein
of practical diet into bodyweight and egg protein
is 55% (Scott

et al

. 1976). The hen needs
bodyweight


0.18


0.5 g protein per 1 g of body-
weight gain at the early stage of egg production. The
daily total of endogenous nitrogen excretion in the
adult chicken, including normal feather loss, could
be expressed as 201 mg per kg bodyweight to the
0.72 power (Harris 1966). The CP requirement for
maintenance is calculated as
201


bodyweight (kg

0.72

)


6.25 of protein/day.
A fresh egg contains 66% water, 12% protein, 10%
fat, 1% carbohydrate and 11% ash (Researches Coun-
cil Science & Technology Agency 2000). Provided that
efciency of protein use is 55%, the protein needed for
1 g of egg is calculated to be 0.66


0.55 g. Sixty grams
of egg contains 7.2 g CP and the efciency of use of
amino acid for egg protein synthesis is 55%, the CP
requirement of laying hens at 100% egg production
rate is estimated to be 16.3 g per day. When 110 g of
diet is consumed, the dietary CP level is calculated to
be 14.8%. For 90, 80, and 70% of egg production, the
required CP levels are 13.3, 11.8, and 10.4%, respec-
tively. These values added to the requirement for
maintenance correspond with those determined by
Okazaki

et al

. (1994).

DECREASE IN NITROGEN EXCRETION
OF LAYING HENS

The scale of livestock production has become inten-
sive, and the number of livestock per farm has
increased in the intensive farms. The amount of feces
and urine excreted by livestock amounts to
92.9 million tons annually, in which 0.7 million tons
nitrogen and 0.5 million tons phosphorus are included
(Takemasa & Takagi 2001). Although there is a large
demand for excreta as composts, the amount of
excreta in intensive farms is exceeding the demand of
excreta as composts locally. In these circumstances,
there is a need to develop a method to decrease not
only nitrogen and phosphorus, but also the total
amount of waste. In the case of phosphorus, it is pos-
sible to reduce phosphorus excretion by supplying
phytase in pigs, broilers and laying hens as summar-
ized by Saito (2001). By reducing dietary nitrogen
level, nitrogen excretion is reduced in broilers (Ishiba-
shi

et al

. 1997), but the excess reduction of dietary
nitrogen level causes the increment of abdominal fat
of broilers.
In practical farms, a laying hen excretes 75 g
excreta in the low oor cage and 100 g excreta in
the high oor cage. As shown in Table 3, when the
diets with 14.020.0% CP diets in which each
essential amino acid was adjusted to be 110145%
of the NRC requirement (1997), were supplied at
the peak egg production stage, excretory nitrogen
(Y, g/hen/day) increased with increasing dietary CP

T. ISHIBASHI and C. YONEMOCHI

Animal Science Journal

(2003) 74, 457469

464

levels (X, % of diet) as follows (Ishibashi

et al

.
1977a):
Y = 0.121X 0.43 (14


20,

R

2

= 0.992).
The retained nitrogen in the body increased
slightly, which might have caused the low accumula-
tion of abdominal fat as observed in broilers (Bartov
& Plavnik 1998) and attribute the renewal of feath-
ers. By adding four enzymes (phytase, protease,
xylase, and cellulose) and ve crystalline amino
acids, Arg, Lys, Met, Trp, and Val to meet 110% of
requirements of Japanese Feeding Standard for Poul-
try (MAFFRC 1998) to the diets, it was possible to
reduce the dietary CP level from 18% of commer-
cial level to 14% without affecting performance of
laying hens and the excretory nitrogen was reduced
drastically by 41% in a practical farm (Ishibashi,
unpubl. data, 2003).
Although there are some differences in the reduc-
tion rate of excretory nitrogen among the reports as
summarized by Saito (2001), it is clear that a large
amount of excretory nitrogen is reduced by decreasing
dietary CP levels in laying hens. In the case of broilers,
the nitrogen excretion was reduced by restricting feed
intake (Ishibashi

et al

. 1977a). By restricting feed
intake, the excreta of laying hens as observed in broil-
ers may be reduced.

QUALITY OF EGGS AND MEAT
OF SPENT HENS
Quality of eggs

In Japan, more than 70% of people believe that the
brown-shelled eggs are more favorable, tasty and
nutritious compared to white-shelled eggs, and eggs
produced in a free range are nutritious and better for
good health compared to those produced in a cage
(Horiguchi

et al

. 1996; Horiguchi 1998). Presently
more than 500 kinds of what are called specied eggs
are on sale, but there are less than 10 kinds of eggs
with the selling point of being more nutritious and
tasty. However, it is not proven that these eggs are
more nutritious and tasty. This fact indicates that it is
difcult to control the contents of taste active compo-
nents of eggs. In a series of sensory test, there were
no differences in the taste of eggs among strains of
hens in Japan, feeding managements, cage and free-
range feeding, and feeds, except that eggs of young
hens were preferred to those of old hens and those of
Silky hens were not preferred to those of Single
Comb White Leghorn hens.
Although eggs of Silky hens were not preferred to
those of Single Comb White Leghorn hens, no differ-
ences were found in the content of taste active com-
ponents, Glu, inosinic acid and other taste active
components among eggs of Silky, Single Comb White
Leghorn and Rhode Island Red hens. When 5% of
Glu was added to the CP 15% diet, the contents of
Glu in neither plasma nor meat increased (Horiguchi
1998). When Met was added to the diets with 16.5%
CP diet consisting of corn and soybean meal, CP and
DM contents of egg white increased, but DM content
of egg yolk and functional components as a food were
not affected (Shafer & Rose 1998). Although the
components of fatty acids and biological active sub-
stances in egg yolk is affected, that in egg white is not
affected by dietary components, feeding management
(Kishii 2002) and breeds of hens (Pobrow & Morris
1974).

Quality of meat of spent hens

In the last 5 years, approximately 89 billion spent hens
weighing 158 kilotons were slaughtered annually, and
more than half of them were rendered to produce

Table 3

Effects of dietary CP on nitrogen excretion of laying hens at 220230 days of age

Diet Dietary CP
(%)
BW change
(g/hen/d)
Feed intake
(g/hen/d)
Nitrogen (mg/hen/d)
Intake Egg Excreta Feather Retention
Experimental 14.0 6.0 100.0 2242

d

955 (42.6)

1

1274

d

(100.0)

2

9 4

d

16.0 9.1 98.9 2532

c

949 (37.5) 1471

c

(115.5) 15 97
c
18.0 6.5 101.7 2929
b
943 (32.2) 1778
b
(139.6) 8 200
b
20.0 10.1 101.5 3428
a
936 (28.8) 1978
a
(155.3) 11 303
a
Commercial 19.8 7.1 99.4 3149
a
968 (30.7) 1905
a
(96.3) 16 270
a
Pooled SE 0.3 2.0 39 18 26 2
Each value is mean for 15 hens with 1680 11 g for initial bodyweight (BW),
ad
Means in the same column with different superscripts
differ signicantly (P < 0.05).
12
Values in parentheses are percentage of nitrogen intake and of the 14.0% crude protein (CP) diet, respectively.
Amino acid nutrition
Animal Science Journal (2003) 74, 457469 465
chicken meal and the remainders were used for meat,
soup and sausage. Egg producers have to pay approx-
imately 020 yen per hen to dealers of spent hens.
These differences in price of spent hens depend on
whether there is a rendering faculty or not. These
expenses have to be lowered or changed to produce
prots. In the case of old dairy cows after the last
breeding, they are fattened for production of meat. In
the case of spent hens, they are all out to alternate to
young pullets, in spite of that, some of them are keep-
ing a high egg production rate. Because there are no
available reports studying the fattening of spent hens,
a new method to improve the cost of spent hens is
required.
ANIMAL WELFARE AND
INDUCED MOLT
The concept of animal welfare is to keep animals in a
comfortable condition. The present poultry produc-
tion system in Japan does not practice good animal
welfare but bad welfare and high production, involv-
ing high population density per cage, keeping birds in
low light intensities, beak trimming, fasting for
induced molt, transport and slaughter. The concept of
animal welfare often conicts with economic and
effective animal production. The main object of nutri-
tion is to optimize productive efciency, but this is
usually achieved only when health is also optimized.
Therefore, health and consequent welfare are major
priorities in modern nutritional practices. The present
egg production system will be improved step-by-step
in future.
In the case of group-fed laying hens, they are de-
beaked at a young age and conned in a narrow cage,
in which excess energy consumption and struggles
with other hens are decreased.
With advancing age, the egg production rate (Fig. 2)
and egg quality decrease. By induced molt, the egg
production rate is recovered to 90% of the maximum
egg production rate before molt. Induced molting is
repeated two or more times. Induced molting is prac-
ticed in more than 50% of farms in the world and is
increased especially when the chick feeding cost is
expensive and egg price is high. The main objective of
induced molting is to improve the quality of eggs and
to extend the productive life of hens. The most popu-
lar method of induced molting involves reducing
bodyweight by fasting for more than a week. Then the
layers are supplied a low CP post-molting diet until
their egg production rate reaches 5%, and then the
post-molting diet is switched to a layer diet. Single
Comb White Leghorn hens loose approximately 30%
of bodyweight (440 g) and 130 g of feather, which are
recovered within 2 weeks after refeeding (Fukuma &
Ishibashi 1997). The CP requirements during this
period were estimated to be less than 14.3% (Fukuma
& Ishibashi 1997), 12.4% (Hoyle & Garlich 1987) and
13.0% (Koelkebeck et al. 1991), respectively, in which
the total SAA were contained 0.56, 0.62 and 0.46%,
respectively.
Because feather contains approximately 2% of Cys
and 0.7% of Met, the requirement of total sulfur con-
taining amino acid (SAA) may be high. The obtained
total SAA requirements from the data of performance
and plasma free Met concentration were estimated to
be 0.540.57% of diet, which were recalculated to be
0.47 and 0.50% on a digestible amino acid basis using
the data by Yamazaki and Kaku (1988). These results
corresponded with those of Carderon and Jensen
(1990) and Schutte and Smink (1998).
PROBLEMS TO BE SOLVED
IN THE FUTURE
The world population has increased annually at rates
ranging between 1.3 and 2.0% since the 1960s. The
general improvement in food consumption habits
throughout the world may prompt the increased con-
sumption of animal products, such as milk, meats and
eggs. Therefore, the challenges of developing effective
production of animal protein will be essential.
Injection of amino acid into hatching eggs
Large chicks are hatched from large eggs. Large chicks
tend to grow faster at hatching time. Administration of
amino acid mixture to hatching eggs increases body-
weight of chicks at hatching time (Ohta et al. 1999,
2001). The injection of nutrients into hatching eggs is
not technically difcult and will be in practice in the
future.
Amino acid requirements of
replacing pullets
It has been a standard practice to feed growing egg-
type pullets a series of diets generally termed starter,
grower, and developer diets. In these rations, CP and
other nutrients decrease. The NRC lists the following
dietary CP requirements for pullets: 18% for 0
6 weeks, 16% for 712 weeks and 15% for 12
18 weeks of age. There are different approaches to
feeding replacement pullets for a number of reasons.
T. ISHIBASHI and C. YONEMOCHI
Animal Science Journal (2003) 74, 457469 466
During the last decade, the poultry industry has been
using pullets that mature earlier. With these birds, it is
of even greater importance that pullets come into pro-
duction at the proper weight and frame size. The more
important factors to be studied for nutritional require-
ments of pullets are concerning the subsequent laying
performance of pullets. There are a few available stud-
ies regarding the effects of CP levels in pullet diets on
development and subsequent layer performance. Mor-
tality and days to 5% egg production, feed intake, feed
conversion and egg weight during the rst 16 weeks
were not affected by rearing dietary treatments (Hus-
sein et al. 1996). It is difcult to conrm the effect of
pullet diet on the subsequent performance, but the
accumulation of more data on the subsequent perfor-
mance is expected.
Improvements of amino acid use
Phytase has been used as a commercial feed additive
for 10 years. In general, the addition of microbial
phytase to the diet improved the digestibility of CP by
13% in addition to positive effect on phosphate
digestibility (Saito 2001). However, the exact mecha-
nisms responsible for improvements remain unclear at
present (Kies et al. 2001). The digestibility of CP in
commercial diets is less than 90%. Therefore, a new
method to improve the digestibility of dietary CP
should be developed by supplementing enzymes, pro-
cessing feedstuffs and so on.
Development of new feedstuffs
The research into new feedstuffs has continued.
Recently new attention has been paid to the re-use of
food waste. The amount of food waste is not major
quantitatively. Importantly, the most attention is
focused on genetically modied plant feedstuffs.
Advances in plant breeding and genetic engineering
are resulting in many genetically altered feedstuffs
that have increased nutritional values for poultry
diets. For example, high oil corn, nutrient dense corn,
high Lys soybean and trypsin-inhibitor and lectin-free
soybean have increased nutritional value for poultry
compared to conventional feedstuffs. Trypsin-inhibitor
and lectin are two major anti-nutritional factors in
soybean. These feedstuffs contain higher levels of
digestible amino acids (Howie 1998).
It has been reported that the share of planted area of
genetically modied plants in 2003 exceed 68% for
soybean and 36% for corn in USA, 63% for rape in
Canada, and 45% for cotton in Australia (Interna-
tional Service for the Acquisition of Agri-biotech
Applications 2003). The safety of these genetic modi-
ed plants on broilers, layers, pigs, and dairy cows is
being studied in our laboratory.
Timing of supply of amino acids
The accumulation rate of protein in egg yolk is con-
stant and it takes 810 days to make egg yolk, but the
synthesis of egg white is not constant and complemen-
tary, and is highest during 3 h before ovulation
(Table 4, Ishibashi & Kumagai 1984; Kumagai & Ish-
ibashi 1984).
When the dietary supply of amino acids is not
enough to meet the temporal high needs of amino
acids, the decient amino acids may be supplied by the
degradation of muscle protein (Hayashi et al. 1991).
The rate of break down was 4.8% per day before ovi-
position and was 8.4% per day after. At these times
the absolute rates of muscle protein break down is cal-
culated to be 1.2 and 2.1 g/h, respectively, which is
enough to meet the temporal requirement for egg
white protein synthesis.
Corticosteroides are known to strongly accelerate
muscle protein breakdown (Beuving & Vonder 1977)
and a maximum blood concentration of corticosterone
had been observed 44 min before oviposition. These
results might support that muscle protein is a temporal
source of amino acids for egg white synthesis.
Egg shell formation is accelerated 1820 h after ovi-
position. The need for Ca increases at night (Horikawa
et al. 1989). By supplying Ca during the night, the egg
shell quality is improved. Like this, by supplying
amino acids when egg white is synthesized, the use of
dietary amino acid for egg white synthesis may be
improved.
CONCLUSION
After the outbreak of bovine spongiform encephalop-
athy encephalitis, the use of protein derived from cat-
tle is prohibited and the use of animal protein is
Table 4 Apparent accumulation rate of soluble protein in the
magnum during egg formulation cycle
Hours after oviposition Accumulation rate (g/h)
02 0.200
25 0.100
510 0.140
1021 0.127
2124 0.447
Amino acid nutrition
Animal Science Journal (2003) 74, 457469 467
avoided. By increasing the use of plant protein, the
volume and moisture of excreta increase and the
importance of amino acids as supplements is increas-
ing. Because the concentration of SAA in an egg is
higher than in the protein derived from corn and soy-
bean meal, SAA is most liable to be limiting and fol-
lowed by Lys, Thr, and Trp in the feed of laying hens.
The addition of Met is commonly in practice. In Japan,
12 amino acids including analogs are permitted as sup-
plements to feeds. The consumption of animal protein
is increasing with an increasing human population
and the improvement of the income levels of the
nation. For effective use of feedstuffs, the use of amino
acid additives is increasing 5% in developed countries
and 10% in underdeveloped countries annually.
Annual production of Lys, Met including DL-Met and
hydroxyanalogs of Met, Thr, and Trp amount to 550,
450, 50, and 0.5 kilotons, respectively. Other amino
acids liable to be limiting will be permitted in near
future. With effective use of these amino acids, feed-
stuffs will be spared, the competition between foods
and feedstuffs will decrease and excretion of waste,
especially nitrogen, will also be decreased.
A wave of free trade would hasten an increased
understanding of animal welfare and hazard analysis
critical control point in the world. Under these circum-
stances, it is expected that the poultry industry will
develop by improving feed efciency with accom-
panying genetic, physiological and management
improvements.
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