Joint FAO/WHO/UNU Expert Consultation on

Energy and Protein Requirements

Rome, 5 to 17 October 1981

ESSENTIAL AMINO ACID REQUIREMENTS: A REVIEW

by

Marian E. Swendseid
University of California
Los Angeles

Most of the studies of essential amino acid requirements with semi-synthetic diets were
conducted during the 1950s and 1960s using nitrogen balance or nitrogen retention as the
criterion of adequacy. Dietary levels of a single amino acid were varied in successive periods
of 5 to 10 days duration. These studies have been reviewed in detail by Irwin and Hegsted
(1). In addition to studies with varying quantities of single amino acids, investigations with
different amounts of complete essential amino acid mixtures or single food proteins of known
amino acid composition and digestibility have also provided information and verification of
essential amino acid requirements. The results of all of these investigations have been
considered and evaluated by both the FAO/WHO Expert Committee on Energy and Protein
Requirements (2) and the Committee on Amino Acids of the Food and Nutrition Board (3). In
this paper new information published since the deliberations of these committees will be
reviewed. Attention will be given to investigations of biochemical indices that might prove
useful in assessment of amino acid requirements and factors which may affect amino acid
requirements will be considered.

Amino Acid Requirements of Infants and Children

The experimental data on amino acid requirements for infants and 10–12-year-old children
have been expanded by Pineda and co-workers (4) to include requirements for the 2-year-old
child. Forty-two healthy children received diets consisting of a core of 0.3 g of cow's milk
protein/kg/day plus an amino acid mixture in proportions and amounts equal to 0.9 g milk
protein/kg/day. Diets provided 100 kcal/kg/day with proper vitamin and mineral supplements.
The single essential amino acid under study was partially replaced in the diet by glycine at
five different levels. Nitrogen balance (4 day periods) was calculated with an allowance of 8
mg N/kg/day for integumental losses. It was assumed that a retention of 16 mg N/kg/day
allowed for normal growth and results were validated with studies conducted in children fed
milk or soy protein to assess protein needs. The following requirements were suggested as
mg/kg/day: isoleucine, 31; lysine, 64; sulfur amino acids, 27; threonine, 37; and tryptophan,
14. The investigators concluded that for children in this age group the FAO/WHO 1973
estimates may be too high for isoleucine, sulfur amino acids, threonine and valine. Fasting
plasma amino acid levels were also measured and it was concluded that reductions in specific
amino acids levels occur in plasma as amino acid intakes are decreased to sub-optimal levels.
Thus, in young children as in infants, fasting plasma amino acid levels appear useful in
estimating amino acid requirements.

Graham, MacLean and co-workers have data in both infants and young children fed soy
protein (5) or wheat protein (6), that the amino acid known to be limiting in these proteins is
decreased in plasma 3 hrs post-prandial in contrast to other amino acids, which are either
increased or stable. Supplementation with methionine for soy and lysine for wheat corrected
this condition. Thus the posprandial plasma amino acid response seems to be helpful in
quantitating amino acid needs in growing children. Fomon (7) has also shown that in infants
fed a soy bean diet when a supplement of methionine was added, weight gain improved and
the serum urea nitrogen level decreased. Hence for infants and young children both amino
acid and urea concentrations in plasma might be employed to gain information on amino acid
requirements.

Amino Acid Requirements of Adults

Evidence is accumulating that histidine is an essential amino acid for adults. It was found that
when uremic patients receiving mixtures of the Rose eight essential amino acids were given a
histidine supplement, hemoglobin increased (8) and nitrogen retention improved (9). Later
Kopple et al. (10) studied 3 normal (ages 48, 38 and 24 yrs) and 3 uremic men (ages 55, 54,
43 yrs) who ingested a histidine-deficient amino acid diet of approximately 6.5 g N/day.
These subjects except for one individual went into negative nitrogen balance after periods of
20 to 30 days. Strong positive nitrogen balance was immediately restored on the addition of
1200 mg of dietary histidine in all but the one subject mentioned previously who was in
negative balance after 5 days of receiving the histidine-deficient diet. In 24 hrs plasma
histidine levels fell to 52% of control values with the 40 g protein diet. By the end of the
histidine-depletion period, plasma histidine levels were 17% of control values. Muscle free
histidine concentrations also decreased. Serum albumin and hematocrits fell and serum iron
rose. Subjects felt unwell and in five subjects a erythematous skin lesion appeared. All
clinical symptoms and skin lesions disappeared with the histidine supplement. Anderson et al.
(11) have also investigated six college-age men consuming for 1 week 6.3 g N amino acid
diets with or without histidine or histidine plus arginine. Mean nitrogen balances (last 4 days
of period) were positive only when histidine was present in the diet. Cho et al. (12) observed
reduced plasma histidine levels when the young men referred to above ingested low-histidine
diets. In further studies of the amounts of histidine that may be required to maintain nitrogen
balance over prolonged time periods, 9 normal and 4 uremic men were fed amino acid diets
containing 6.5 g N and 4, 8, or 12 mg/kg of histidine for 27 ± 5 days per period (13). The
average age of subjects was 44 years. Fasting plasma histidine levels rose with increasing
histidine intake and 1 hr postprandial levels were higher only in subjects ingesting 12 mg
histidine/kg. Likewise the uptake of radio-iron into red cells was lower than the normal range
for all subjects ingesting 4 mg histidine/kg, lower in 3 of 6 subjects receiving 8 mg/kg and
within the range for 5 subjects fed 12 mg/kg. From these data, it appears that the dietary
histidine requirement in both normal and uremic men is more than 8 mg/kg/day and may be
as high as 12 mg/kg/day. For a 70 kg man the range is from 560 to 840 mg of histidine per
day.

These observations indicate that histidine is unique among essential amino acids in that short-
term nitrogen equilibrium is maintained with extremely low histidine-containing diets. There
are several hypotheses to explain this phenomenon: 1) histidine comprises eight percent of
the hemoglobin molecule and the breakdown of hemoglobin contributes more histidine in
proportion to other essential amino acids; 2) the hydrolysis of the dipeptide carnosine from
muscle releases histidine. There is good evidence that in rats and chicks the muscle carnosine
content is decreased with histidine-free diet and replenished with histidine supplementation
(14, 15) and 3) there is some biosynthesis of histidine (16). A combination of these
hypotheses may provide the explanation for the unique response of body tissues to the dietary
removal of histidine. No reports of histidine requirements of women have been found.
However, it is possible that in women fed histidine-deficient diets negative nitrogen balance
might develop more rapidly than in men because of a decreased muscle mass and thus
perhaps more limited carnosine stores.

Young and co-workers have presented additional data on valine, lysine (17), tryptophan (18,
19) and threonine requirements (20) using nitrogen balance and the plasma amino acid
response curve. In human subjects, for some amino acids at least, as dietary intake is
increased, plasma levels remain stable for a time, then rise abruptly to be followed by another
plateau. Young has attempted to quantitate the amino acid intake at the point where plasma
levels begin to increase, the “breakpoint” and compare this intake level with the amount
needed to maintain nitrogen equilibrium. These investigators have also used postprandial
amino acid levels as indicators of dietary requirements. They have found that in general the
lower “breakpoint” of the plasma amino acid response curve corresponds to the minimum
levels of amino acid intake that is necessary to maintain nitrogen balance. It is possible
therefore in the case of essential amino acids with plasma levels responsive to dietary intake
that criteria based on plasma amino acid concentrations can be developed to assess
requirements.

The investigations of Young and co-workers have included elderly subjects and have
provided new data on specific essential amino acid requirements for subjects in this age
group. When subjects were ingesting 0.5 g protein/kg body wt, the mean minimum
requirement of elderly people for tryptophan was 2 mg/kg body weight/day and for threonine
it was 8 mg/kg. These values are similar to those estimated for young subjects.

Plasma response curves have also been used in an attempt to evaluate histidine requirements
(21). Five normal and two uremic men received amino acid diets in which histidine intakes
were varied from 60 to 2800 mg/ day at 8-day intervals. Postabsorption plasma and urinary
histidine levels were correlated with histidine intake but the plasma response curve did not
demonstrate a consistent breakpoint which could be used to indicate histidine requirement.
When 6 normal subjects were fed 2 mg/kg/day or less, nitrogen balance (not corrected, for
integumental losses) was -0.18 ± 0.5 mg/kg/day, suggesting that histidine requirements are
greater than this amount.

Summary of Current Evidence on Amino Acid Requirements:

The conclusions of the reviewers of amino acid requirement studies in 1971 (1) appear to be
quite relevant even today. Data are still lacking in children of some age groups and in women
during pregnancy and lactation. The number of adult subjects who have been studied in terms
of needs for specific essential amino acids is small and there is an apparent marked variability
among individuals. Also, differences are great between study designs. The NRC/FN
Committee on Amino Acids (22) has indicated that with present criteria of estimation, it
seems unlikely that there is a difference in essential amino acid requirements expressed per
kilogram of body weight between men and women. This conclusion would appear to be still
valid.

It would appear that although there are some conflicting data, the preponderance of studies
suggests that essential amino acid requirements of the elderly do not differ from younger age
groups, at least when the total nitrogen intake is low.
Factors to be Considered in Assessing Essential Amino Acid Requirements:

It is becoming increasingly apparent that a knowledge of essential amino acid requirements is

of great practical value for evaluating food supplies and instituting supplementation when it
is needed. It would appear that a considerable expansion of the amino acid requirement
studies is a necessary prerequisite for obtaining a valid data base and factors to be considered
in designing future investigations should be carefully evaluated.

Energy intake: Recent studies of protein requirements have clarified the quantitative
relationships between total nitrogen and energy intake (23, 24). Calloway (24) has found that
68 ± 15 mg/kg of egg N maintained nitrogen balance and weight in young men with a dietary
intake of 43 ± 4.4 kcal/kg but with an intake of 39.6 ± 4.4 kcal, 89 ± 18 mg N per kg was
required. A slight adjustment in energy intake with marginal N intake can substantially
perturb N balance. It has been often stated that amino acid requirements are based on dietary
energy contents that in many instances were higher than the amount required to maintain
weight and were above the usual intake. In the ideal study of amino acid needs, weight and
body composition should be maintained.

Length of study period: Amino acid requirements were formulated on the basis of short-term
nitrogen balance studies. However protein allowances sufficient to promote short-term
nitrogen balance have now been found to be inadequate to maintain long-term nitrogen
balance and body potassium content (25). Although there is evidence that the limiting
nitrogen component in these studies is non-protein nitrogen (26) these results bring into focus
the possibility that the minimum or “safe” amount of one or more of the essential amino acids
also may not be sufficient for prolonged periods. It is now known that dietary histidine
inadequacy affects nitrogen balance only after a period of 15–20 days or longer (vide
infra)(10).

Total nitrogen intake: There is now good evidence, including the study referred to above (26)
that the limiting nitrogen component in high quality proteins is not the essential but rather the
nonessential amino acids (27, 28). When nonessential nitrogen is added to high quality
protein, nitrogen balance is promoted. There are also other circumstances related to
nonessential nitrogen additions where nitrogen balance could be enhanced. When energy
intake is limited, additional nonessential nitrogen through provision of extra calories could
increase nitrogen retention. It is also possible that extra nonessential nitrogen may increase
requirements for essential amino acids. In older individuals there is limited evidence that the
requirement for an essential amino acid mixture increases as the total nitrogen intake is
increased from 7 g to 10 g/day and the requirement lessened as total nitrogen intake is
reduced from 7 g to 3.5 g/day (29). It is also possible that the oxidation rate of certain amino
acids may be affected by protein intake. Limited evidence with injection of labeled tyrosine
indicates less expired 14CO2 and therefore decreased oxidation in uremic men fed 20 as
compared to 40 or 60 g protein diets (30). Enhanced oxidation of amino acids could also be a
factor in increasing amino acid requirements with higher protein intakes.

Labile body protein: There is firm information that the protein content of some organs such
as liver, pancreas and gut respond rapidly to changes in protein intake (reviewed by Munro,
31). The amount of nitrogen lost in the urine during the first few days of ingesting a protein-
free diet is considered to represent the labile protein reserve which accumulates when dietary
protein is high and recedes when protein intake is lowered. Munro estimates the capacity of
the human body to accumulate labile protein as 300–400 g. Maintaining this labile protein
must represent a metabolic cost of extra protein and extra energy. When subjects are given
protein-free diets prior to test treatments with amino acid mixtures or when subjects are in
negative nitrogen balance for prolonged periods of time, the labile protein of the body is
depleted and as a consequence it can be presumed that the amino acid requirement would be
decreased. In the study by Young on tryptophan requirements (19), when the dietary
tryptophan intake was 4 mg/kg/day, 6 subjects who had received protein-free diets for 2 days
were in positive nitrogen balance and 8 subjects who had not been given the protein-free diet
were in negative nitrogen balance. In future studies of amino acid requirements more
attention should be given to the state of body labile protein stores. In addition to the amount
of nonessential nitrogen supplied to maintain these stores it is possible the kind of non-
essential nitrogen has some influence (32). The limited evidence that older individuals might
have increased essential amino acid requirements (30, 33) was obtained only in studies with
subjects receiving diets of 7 or 15 g of nitrogen. Since each diet trial period was interspersed
with an isonitrogenous period of ordinary food, labile body protein stores would appear to
have been well-maintained.

Methods of assessment: The limitations of the nitrogen balance method have been reviewed
many times and the recent results of long-term studies have made it clear that this method
cannot be the sole criterion for estimating amino acid requirements in adult subjects. Thus
other methods must be introduced and validated to accurately assess essential amino acid
needs. There is quite good evidence that in infants and children plasma amino acid levels are
responsive to dietary intake and can indicate amino acid deficiencies. Graham and co-
workers (5, 6) are actively exploring the usefulness of both fasting and postprandial amino
acid levels in relation to estimating limiting amino acid needs when various vegetable
proteins are fed. In adult subjects, Young and co-workers are investigating the plasma acid
response curve as described above (17, 18, 19, 20) and also postprandial amino acid levels as
indicators of amino acid needs. These would appear to be methods of promise for evaluating
amino acid requirements.

In the development of new approaches for evaluating essential amino acid requirements,
perhaps there can be methods appropriate for only a single amino acid which can be based on
a specific metabolic need for this amino acid.

The usefulness of alterations in some of the blood proteins with short half lives such as
transferrin and retinol binding protein should be explored. The sophisticated studies of Young
and Bier and co-workers using infusions of stable isotope-labeled amino acids to measure
turnover may provide the ultimate approach to assessing amino acid requirements with
different dietary conditions (34, 35).

Requirement for histidine: It has been established that short-term nitrogen balance studies are
not useful in assessing daily histidine needs (10, 11). The daily requirement estimates of 8 to
12 mg/kg are based on results obtained from labeled iron uptake into red cells and the data
have not been published in detail. The true histidine requirement would appear to consist of
maintaining not only body protein but also carnosine stores. The response curve of plasma
histidine to dietary histidine did not demonstrate a consistent breakpoint that could be useful
in indicating the histidine requirement (21). However both postabsorptive plasma histidine
and urinary histidine levels are correlated with dietary histidine intake (21) and it would
appear that these parameters might ultimately prove useful in evaluating histidine
requirements.
Sebagian besar studi tentang persyaratan asam amino esensial dengan diet semi sintetis dilakukan
selama tahun 1950an dan 1960an menggunakan keseimbangan nitrogen dan retensi nitrogen
sebagai kriteria kecukupan. Tingkat diet asam amino tunggal bervariasi dalam periode 5 sampai 10
hari berturut-turut. Penelitian ini telah diulas secara rinci oleh Irwin dan Hegsted (1). Selain
penelitian dengan jumlah asam amino tunggal yang bervariasi, penyidikan dengan jumlah yang
berbeda dari campuran asam amino esensial lengkap atau protein makanan tunggal dari komposisi
asam amino yang diketahui dan kecernaan juga telah memberikan informasi dan verifikasi
persyaratan asam amino esensial. Hasil dari semua investigasi ini telah dipertimbangkan dan
dievaluasi oleh FAO / WHO Expert Committee on Energy and Protein Requirements (2) dan Komite
Asam Amino dari Dewan Makanan dan Gizi (3). Dalam makalah ini muncul informasi baru sejak
pembahasan panitia tersebut akan ditinjau ulang. Perhatian akan diberikan pada penyelidikan indeks
biokimia yang mungkin berguna dalam menilai persyaratan asam amino dan faktor-faktor yang
dapat mempengaruhi persyaratan asam amino akan dipertimbangkan.

Persyaratan Asam Amino untuk Bayi dan Anak

Data eksperimental mengenai kebutuhan asam amino untuk bayi dan anak usia 10-12 tahun telah
diperluas oleh Pineda dan rekan kerja (4) untuk memasukkan persyaratan untuk anak berusia 2
tahun. Empat puluh dua anak sehat menerima makanan yang terdiri dari inti 0,3 g protein susu sapi /
kg / hari ditambah campuran asam amino dalam proporsi dan jumlahnya sama dengan 0,9 g protein
susu / kg / hari. Diet menyediakan 100 kkal / kg / hari dengan suplemen vitamin dan mineral yang
tepat. Asam amino esensial tunggal yang diteliti sebagian diganti dalam makanan dengan glisin pada
lima tingkat yang berbeda. Keseimbangan nitrogen (4 hari menstruasi) dihitung dengan penyisihan 8
mg N / kg / hari untuk kerugian integumental. Diasumsikan bahwa retensi 16 mg N / kg / hari
diperbolehkan untuk pertumbuhan normal dan hasilnya divalidasi dengan penelitian yang dilakukan
pada anak-anak yang diberi susu atau protein kedelai untuk menilai kebutuhan protein. Persyaratan
berikut disarankan sebagai mg / kg / hari: isoleusin, 31; Lisin, 64; Asam amino sulfur, 27; Threonine,
37; Dan triptofan, 14. Para peneliti menyimpulkan bahwa untuk anak-anak di kelompok usia ini
perkiraan FAO / WHO 1973 mungkin terlalu tinggi untuk isoleucine, asam amino belerang, treonin
dan valin. Kadar asam amino plasma puasa juga diukur dan disimpulkan bahwa penurunan kadar
asam amino spesifik terjadi pada plasma karena asupan asam amino menurun sampai tingkat
suboptimal. Jadi, pada anak kecil seperti pada bayi, kadar asam amino plasma puasa tampak
berguna dalam memperkirakan kebutuhan asam amino.

Graham, MacLean dan rekan kerja memiliki data pada bayi dan anak-anak yang diberi protein
kedelai (5) atau protein gandum (6), bahwa asam amino yang diketahui membatasi protein ini
mengalami penurunan pada plasma 3 jam setelah prandial kontras. Ke asam amino lain, yang
meningkat atau stabil. Suplementasi dengan metionin untuk kedelai dan lisin untuk gandum
memperbaiki kondisi ini. Dengan demikian respons asam amino plasma posprandial tampaknya
membantu dalam mengkuantifikasi kebutuhan asam amino pada anak yang sedang tumbuh. Fomon
(7) juga menunjukkan bahwa pada bayi mengonsumsi makanan kacang kedelai saat suplemen
metionin ditambahkan, kenaikan berat badan meningkat dan kadar nitrogen urea serum menurun.
Oleh karena itu untuk bayi dan anak-anak, baik konsentrasi asam amino dan urea dalam plasma
dapat digunakan untuk mendapatkan informasi tentang persyaratan asam amino.
Persyaratan Asam Amino Orang Dewasa

Bukti terakumulasi bahwa histidin merupakan asam amino esensial untuk orang dewasa. Ditemukan
bahwa ketika pasien uremik yang menerima campuran dari Rose delapan asam amino esensial diberi
suplemen histidin, hemoglobin meningkat (8) dan retensi nitrogen meningkat (9). Kemudian Kopple
dkk. (10) mempelajari 3 orang normal (usia 48, 38 dan 24 tahun) dan 3 pria uremik (usia 55, 54, 43
thn) yang menelan diet asam amino histidin kurang dari 6,5 g N / hari. Subjek ini kecuali satu individu
mengalami keseimbangan nitrogen negatif setelah periode 20 sampai 30 hari. Keseimbangan
nitrogen positif yang kuat segera dipulihkan pada penambahan 1200 mg histidin diet dalam semua
hal kecuali satu subjek yang disebutkan sebelumnya yang berada dalam keseimbangan negatif
setelah 5 hari menerima diet kekurangan histidin. Pada 24 jam kadar histidin plasma turun menjadi
52% nilai kontrol dengan diet protein 40 g. Pada akhir periode histidin-deplesi, kadar histidin plasma
adalah 17% dari nilai kontrol. Konsentrasi histidin bebas otot juga menurun. Albumin serum dan
hematokrit turun dan besi serum naik. Subjek merasa tidak sehat dan dalam lima subjek lesi kulit
eritematosa muncul. Semua gejala klinis dan lesi kulit hilang dengan suplemen histidin. Anderson
dkk. (11) juga telah menyelidiki enam pria usia kuliah yang mengkonsumsi 1 minggu 6,3 g diet asam
amino N dengan atau dengan Histidin atau histidin plus arginin. Rata-rata saldo nitrogen (4 hari
terakhir) hanya positif bila histidin hadir dalam makanan. Cho et al. (12) mengamati kadar histidin
plasma berkurang saat orang muda merujuk pada diet histidin rendah yang tertelan. Dalam studi
lebih lanjut tentang jumlah histidin yang mungkin diperlukan untuk menjaga keseimbangan nitrogen
selama periode waktu yang lama, 9 pria normal dan 4 orang uremik diberi makanan asam amino
yang mengandung 6,5 g N dan 4, 8, atau 12 mg / kg histidin selama 27 ± 5 hari per periode (13). Usia
rata-rata subjek adalah 44 tahun. Tingkat histidin plasma puasa meningkat dengan meningkatnya
asupan histidin dan tingkat postprandial 1 jam lebih tinggi hanya pada subyek yang mengkonsumsi
12 mg histidin / kg. Demikian pula serapan radio-besi ke dalam sel darah merah lebih rendah dari
pada kisaran normal untuk semua subyek yang menelan 4 mg histidin / kg, lebih rendah pada 3 dari
6 subjek yang mendapat 8 mg / kg dan dalam kisaran untuk 5 subjek diberi makan 12 mg / kg. Dari
data tersebut, tampak bahwa kebutuhan histidin diet pada pria normal dan pria uremik lebih dari 8
mg / kg / hari dan mungkin setinggi 12 mg / kg / hari. Untuk pria 70 kg, kisarannya berkisar antara
560 sampai 840 mg histidin per hari.

Pengamatan ini menunjukkan bahwa histidin unik di antara asam amino esensial dalam ekuilibrium
nitrogen jangka pendek dipertahankan dengan diet histidin yang sangat rendah. Ada beberapa
hipotesis untuk menjelaskan fenomena ini: 1) histidin terdiri dari delapan persen molekul
hemoglobin dan pemecahan hemoglobin menyumbang lebih banyak histidin sebanding dengan
asam amino esensial lainnya; 2) hidrolisis carvoine dipeptida dari otot melepaskan histidin. Ada bukti
bagus bahwa pada tikus dan anak ayam, kandungan otot carnosine menurun dengan diet bebas
histamin dan ditambah dengan suplementasi histidin (14, 15) dan 3) ada beberapa biosintesis
histidin (16). Kombinasi dari hipotesis ini dapat memberikan penjelasan untuk respon unik jaringan
tubuh terhadap penghapusan histidin. Tidak ada laporan tentang persyaratan histidin wanita yang
ditemukan. Namun, ada kemungkinan bahwa pada wanita yang diberi makanan seimbang histidin,
keseimbangan nitrogen negatif bisa berkembang lebih cepat daripada pada pria karena massa otot
menurun dan oleh karena itu mungkin toko carnosine lebih terbatas.

Young dan rekan kerja telah menyajikan data tambahan tentang valin, lisin (17), triptofan (18, 19)
dan persyaratan treonin (20) menggunakan keseimbangan nitrogen dan kurva respons asam amino
plasma. Pada subyek manusia, untuk beberapa asam amino setidaknya, karena asupan makanan
meningkat, kadar plasma tetap stabil untuk sementara waktu, kemudian naik tiba-tiba diikuti oleh
dataran tinggi lain. Young telah mencoba untuk menghitung asupan asam amino pada titik di mana
tingkat plasma mulai meningkat, "breakpoint" dan bandingkan tingkat asupan ini dengan jumlah
yang dibutuhkan untuk menjaga keseimbangan nitrogen. Penyidik ini juga menggunakan asam
amino postprandial sebagai indikator kebutuhan diet. Mereka telah menemukan bahwa secara
umum 'breakpoint' yang lebih rendah dari kurva respons asam amino plasma sesuai dengan tingkat
minimum asupan asam amino yang diperlukan untuk menjaga keseimbangan nitrogen. Oleh karena
itu, mungkin terjadi asam amino esensial dengan kadar plasma yang responsif terhadap asupan
makanan sehingga kriteria berdasarkan konsentrasi asam amino plasma dapat dikembangkan untuk
menilai kebutuhan.

Penyelidikan Young dan rekan kerja telah mencakup subyek lansia dan telah menyediakan data baru
mengenai persyaratan asam amino esensial spesifik untuk subjek dalam kelompok usia ini. Ketika
subyek menelan 0,5 g protein / kg berat badan, rata-rata kebutuhan minimum orang tua untuk
triptofan adalah 2 mg / kg berat badan / hari dan untuk treonin 8 mg / kg. Nilai ini serupa dengan
yang diperkirakan untuk subjek muda.

Kurva respon plasma juga telah digunakan untuk mengevaluasi kebutuhan histidin (21). Lima pria
normal dan dua orang uremik menerima diet asam amino di mana asupan histidin bervariasi dari 60
sampai 2800 mg / hari pada interval 8 hari. Plasma postabsorpsi dan kadar histidin urin berkorelasi
dengan asupan histidin namun kurva respons plasma tidak menunjukkan breakpoint yang konsisten
yang dapat digunakan untuk menunjukkan kebutuhan histidin. Bila 6 subyek normal diberi makan 2
mg / kg / hari atau kurang, keseimbangan nitrogen (tidak dikoreksi, untuk kerugian integumental)
adalah -0,18 ± 0,5 mg / kg / hari, menunjukkan bahwa persyaratan histidin lebih besar dari jumlah
ini.

Ringkasan Bukti Saat Ini tentang Persyaratan Asam Amino:

Kesimpulan dari penelaah studi kebutuhan asam amino pada tahun 1971 (1) tampaknya cukup
relevan bahkan sampai hari ini. Data masih kurang pada anak-anak dari beberapa kelompok usia dan
pada wanita selama kehamilan dan menyusui. Jumlah subjek dewasa yang telah dipelajari dalam hal
kebutuhan asam amino esensial tertentu kecil dan ada variabilitas yang nyata di antara individu.
Selain itu, perbedaannya bagus antara desain studi. NRC / FN Komite Asam Amino (22) telah
mengindikasikan bahwa dengan kriteria perkiraan saat ini, nampaknya tidak mungkin ada perbedaan
persyaratan asam amino esensial yang dinyatakan per kilogram berat badan antara pria dan wanita.
Kesimpulan ini tampaknya masih berlaku. Akan terlihat bahwa walaupun ada beberapa data yang
bertentangan, semakin banyak penelitian menunjukkan bahwa kebutuhan asam amino esensial
pada lansia tidak berbeda dengan kelompok usia muda, setidaknya bila asupan nitrogen total
rendah. .Faktor yang Harus Dipertimbangkan dalam Menilai Kebutuhan Asam Amino Esensial:
Semakin jelas bahwa pengetahuan tentang persyaratan asam amino esensial adalah nilai praktis
yang bagus untuk mengevaluasi persediaan makanan dan memberi suplemen bila diperlukan. Akan
terlihat bahwa perluasan yang cukup besar dari studi kebutuhan asam amino adalah prasyarat yang
diperlukan untuk mendapatkan basis data yang valid dan faktor-faktor yang harus dipertimbangkan
dalam merancang penyelidikan di masa depan harus dievaluasi secara hati-hati. Asupan energi: Studi
terbaru mengenai persyaratan protein telah mengklarifikasi hubungan kuantitatif. Antara total
asupan nitrogen dan energi (23, 24). Calloway (24) menemukan bahwa 68 ± 15 mg / kg telur N
mempertahankan keseimbangan dan berat nitrogen pada pria muda dengan asupan makanan 43 ±
4,4 kkal / kg namun dengan asupan 39,6 ± 4,4 kkal, 89 ± 18 mg N Per kg diperlukan. Sedikit
penyesuaian asupan energi dengan asupan N marjinal dapat secara substansial mengganggu
keseimbangan N. Telah sering dinyatakan bahwa kebutuhan asam amino didasarkan pada
kandungan energi makanan yang dalam banyak kasus lebih tinggi dari jumlah yang dibutuhkan untuk
mempertahankan berat badan dan berada di atas asupan biasa. Dalam studi ideal kebutuhan asam
amino, berat dan komposisi tubuh harus dipertahankan. Panjang masa studi: Persyaratan asam
amino diformulasikan berdasarkan studi keseimbangan nitrogen jangka pendek. Namun, tunjangan
protein yang cukup untuk meningkatkan keseimbangan nitrogen jangka pendek kini tidak memadai
untuk menjaga keseimbangan nitrogen dan kandungan potassium tubuh jangka panjang (25).
Meskipun ada bukti bahwa komponen nitrogen yang membatasi dalam penelitian ini adalah
nitrogen non-protein (26) hasil ini memusatkan pada kemungkinan bahwa jumlah minimum atau
"aman" dari satu atau lebih asam amino esensial juga mungkin tidak cukup untuk Periode yang
panjang. Sekarang diketahui bahwa kekurangan histidin diet mempengaruhi keseimbangan nitrogen
hanya setelah jangka waktu 15-20 hari atau lebih lama (vide infra) (10). Asupan nitrogen total: Saat
ini ada bukti bagus, termasuk penelitian yang disebut di atas (26) bahwa Komponen nitrogen yang
membatasi protein berkualitas tinggi bukan asam amino esensial tapi agak tidak penting (27, 28).
Bila nitrogen yang tidak penting ditambahkan ke protein berkualitas tinggi, keseimbangan nitrogen
dipromosikan. Ada juga keadaan lain yang terkait dengan penambahan nitrogen yang tidak penting
dimana keseimbangan nitrogen dapat ditingkatkan. Bila asupan energi terbatas, nitrogen tak penting
tambahan melalui pemberian kalori ekstra dapat meningkatkan retensi nitrogen. Ada kemungkinan
juga bahwa nitrogen tak penting tambahan dapat meningkatkan kebutuhan asam amino esensial.
Pada orang tua ada bukti terbatas bahwa persyaratan untuk campuran asam amino esensial
meningkat saat asupan nitrogen meningkat dari 7 g sampai 10 g / hari dan kebutuhan berkurang
karena asupan nitrogen total berkurang dari 7 g menjadi 3,5 g / hari. (29). Mungkin juga tingkat
oksidasi asam amino tertentu mungkin dipengaruhi oleh asupan protein. Bukti terbatas dengan
injeksi tirosin berlabel menunjukkan 14CO2 yang kadaluarsa dan karena itu menurunkan oksidasi
pada pria uremik yang diberi makan 20 dibandingkan dengan 40 atau 60 g protein diet (30). Oksidasi
asam amino yang disempurnakan juga bisa menjadi faktor dalam meningkatkan kebutuhan asam
amino dengan protein yang lebih tinggi. Protein tubuh yang lembut: Ada informasi kuat bahwa
kandungan protein beberapa organ seperti hati, pankreas dan usus merespon dengan cepat
terhadap perubahan asupan protein. Ditinjau oleh Munro, 31). Jumlah nitrogen yang hilang dalam
urin selama beberapa hari pertama menelan makanan bebas protein dianggap mewakili cadangan
protein labil yang terakumulasi saat protein diet tinggi dan surut saat asupan protein diturunkan.
Munro memperkirakan kapasitas tubuh manusia untuk mengakumulasi protein labil seperti 300-400
g. Mempertahankan protein labil ini harus mewakili biaya metabolisme protein ekstra dan energi
ekstra. Bila subjek diberi diet bebas protein sebelum melakukan uji perlakuan dengan campuran
asam amino atau bila subjek berada dalam keseimbangan nitrogen negatif untuk jangka waktu lama,
protein labil tubuh terkuras dan sebagai konsekuensinya dapat diasumsikan bahwa asam amino
Persyaratan akan berkurang. Dalam penelitian oleh Young mengenai triptofan (19), ketika asupan
tryptophan diet adalah 4 mg / kg / hari, 6 orang yang telah menerima makanan bebas protein
selama 2 hari berada dalam keseimbangan nitrogen positif dan 8 subyek yang tidak diberi diet
bebas protein berada dalam keseimbangan nitrogen negatif. Dalam studi selanjutnya tentang
kebutuhan asam amino, perhatian lebih harus diberikan pada keadaan penyimpanan protein
labil tubuh. Selain jumlah nitrogen yang tidak penting yang dipasok untuk memelihara toko
ini, kemungkinan jenis nitrogen non-esensial memiliki beberapa pengaruh (32). Bukti
terbatas bahwa orang yang lebih tua mungkin telah meningkatkan kebutuhan asam amino
esensial (30, 33) diperoleh hanya dalam penelitian dengan subjek yang menerima makanan 7
atau 15 g nitrogen. Karena setiap periode percobaan diet diselingi dengan periode makanan
biasa isonitrogen, toko protein tubuh labil tampaknya sudah terpelihara dengan baik. Metode
penilaian: Keterbatasan metode keseimbangan nitrogen telah ditinjau berkali-kali dan hasil
akhir-akhir ini sangat lama. - Studi kasus telah memperjelas bahwa metode ini tidak dapat
menjadi satu-satunya kriteria untuk memperkirakan kebutuhan asam amino pada subjek
dewasa. Dengan demikian metode lain harus diperkenalkan dan divalidasi untuk menilai
kebutuhan asam amino esensial secara akurat. Ada bukti yang cukup bagus bahwa pada bayi
dan anak-anak kadar asam amino plasma responsif terhadap asupan makanan dan dapat
mengindikasikan kekurangan asam amino. Graham dan rekan kerja (5, 6) secara aktif
mengeksplorasi kegunaan kadar asam amino puasa dan postprandial dalam kaitannya dengan
memperkirakan kebutuhan asam amino yang membatasi saat berbagai protein nabati diberi
makan. Pada subjek dewasa, Young dan rekan kerja menyelidiki kurva respons asam plasma
seperti yang dijelaskan di atas (17, 18, 19, 20) dan juga asam amino postprandial sebagai
indikator kebutuhan asam amino. Ini akan menjadi metode janji untuk mengevaluasi
persyaratan asam amino. Dalam pengembangan pendekatan baru untuk mengevaluasi
persyaratan asam amino esensial, mungkin ada metode yang tepat untuk hanya satu asam
amino yang dapat didasarkan pada kebutuhan metabolik tertentu untuk hal ini. Asam amino.
Kegunaan perubahan beberapa protein darah dengan paruh pendek seperti protein pengikat
transferin dan retinol harus dieksplorasi. Studi canggih Young and Bier dan rekan kerja yang
menggunakan infus asam amino berlabel isotop yang stabil untuk mengukur omset dapat
memberikan pendekatan akhir untuk menilai kebutuhan asam amino dengan kondisi diet
yang berbeda (34, 35). Persyaratan untuk histidin: Telah terjadi Menetapkan bahwa studi
keseimbangan nitrogen jangka pendek tidak berguna dalam menilai kebutuhan histidin
sehari-hari (10, 11). Perkiraan kebutuhan harian 8 sampai 12 mg / kg didasarkan pada hasil
yang diperoleh dari pengambilan besi berlabel ke dalam sel darah merah dan data belum
dipublikasikan secara rinci. Kebutuhan histidin sejati tampaknya terdiri dari pemeliharaan
protein tubuh tidak hanya tetapi juga toko carnosine. Kurva respons histidin plasma terhadap
histidin diet tidak menunjukkan breakpoint yang konsisten yang dapat berguna untuk
menunjukkan kebutuhan histidin (21). Namun histidin histidin plasma postabsorptif dan
kadar histidin urin berkorelasi dengan asupan histidin diet (21) dan tampaknya parameter ini
pada akhirnya terbukti berguna dalam mengevaluasi kebutuhan histidin.
2. ESSENTIAL NUTRIENTS - PROTEINS
AND AMINO ACIDS
2.1 Proteins
The proteins are among the most important constituents of all living cells and represent the
largest chemical group in the animal body, with the exception of water; the whole fish carcass
contains on average 75% water, 16% protein, 6% lipid, and 3% ash. Proteins are essential
components of both the cell nucleus and cell protoplasm, and accordingly account for the
bulk of the muscle tissues, internal organs, brain, nerves and skin.

2.1.1 Composition

Proteins are very complex organic compounds of high molecular weight. In common with
carbohydrates and lipids, they contain carbon (C), hydrogen (H), and oxygen (O), but in
addition also contain about 16 % nitrogen (N: range 12–19%), and sometimes phosphorus (P)
and sulphur (S).

2.1.2 Structure

Proteins differ from other biologically important macromolecules such as carbohydrates and
lipids in their basic structure. For example, in contrast to the basic structure of carbohydrates
and lipids, which is often composed of identical or very similar repeating units (ie. the
glucose repeating unit within starch, glycogen and cellulose), proteins may have up to 100
different basic units (amino acids). It follows therefore that greater compound variabilities
and ranges are possible, not only to composition, but also to protein shape.

2.1.3 Chemical properties

Colloidal in nature, proteins vary in their solubility in water, from insoluble keratin to the
highly soluble albumins. All proteins can be ‘denatured’ by heat, strong acid, alkali, alcohol,
acetone, urea and by heavy metal salts. When proteins are denatured they loose their unique
structure, and therefore possess different chemical, physical and biological properties (ie.
inactivation of enzymes by heat).

2.1.4 Classification

Proteins maŷ be classified into three main groups according to their shape, solubility and
chemical composition:

a. Fibrous proteins: insoluble animal proteins which are generally very resistant to
digestive enzyme breakdown. Fibrous proteins exist as elongated filamentous chains.
Examples of fibrous proteins include the collagens (main protein of connective
tissue), elastin (present in elastic tissues such as arteries and tendons), and keratin
(present in hair, nails, wool and hooves of mammals).
b. Globular proteins: include all enzymes, antigens and hormone proteins. Globular
proteins can be further subdivided into albumins (water soluble, heat-coagulable
 proteins which occur in eggs, milk, blood and many plants); globulins (insoluble or
sparingly soluble in water, and present in eggs, milk, and blood, and serve as the main
protein reserve in plant seeds); and histones (basic proteins of low molecular weight,
water soluble, occur in the cell nucleus associated with deoxyribonucleic acid -
DNA).
c. Conjugated proteins: these are proteins which yield non-protein groups as well as
amino acids on hydrolysis. Examples include the phosphoproteins (casein of milk,
phosvitin of egg yolk), glycoproteins (mucous secretions), lipoproteins (cell
membranes), chromoproteins (haemoglobin, haemocyanin, cytochrome,
flavoproteins), and nucleoproteins (combination of proteins with nucleic acids present
in the cell nucleus).

2.2 Protein function

The function of proteins may be summarised as follows:

 To repair worn or wasted tissue (tissue repair and maintenance) and to rebuild new
tissue (as new protein and growth).
 Dietary protein may be catabolized as a source of energy, or may serve as a substrate
for the formation of tissue carbohydrates or lipids.
 Dietary protein is required within the animal body for the formation of hormones,
enzymes and a wide variety of other biologically important substances such as
antibodies and haemoglobin.

2.3 Protein requirements

The study of dietary nutrient requirements in fishes and shrimp has been almost entirely
based on studies comparable to those conducted with terrestrial farm animals. It follows
therefore that almost all the available information on the dietary nutrient requirements of
aquaculture species is derived from laboratory based feeding trials; the animals being kept in
a controlled environment at high density and having no access to natural food organisms.

2.3.1 Optimum dietary protein level

Based on feeding techniques pioneered and developed for terrestrial animals the dietary
protein requirements of fish were first investigated in the Chinnok salmon (Oncorhynchus
tshawytscha) by Delong, et al, (1958). Fish were fed a balanced diet containing graded levels
of a high quality protein (casein:gelatin mixture supplemented with crystalline amino acids to
simulate the amino acid profile of whole hen's egg protein) over a 10-week period and the
observed protein level giving optimum growth was taken as the requirement (Fig. 2). Since
these early studies the approach used by workers today has changed very little if at all, with
the possible exception of the use by some researchers of maximum tissue protein retention or
nitrogen balance in preference to weight gain as the criterion of requirement (Ogino, 1980).
Dietary protein requirements are normally expressed in terms of a fixed dietary percentage or
as a ratio of protein to dietary energy.
Fig. 2. Typical dose response curve

To date over 30 fish and shrimp species have been examined in this manner and the results
show a uniformly high dietary protein requirement in the range 24–57%, or equivalent to 30–
70% of the gross energy content of the diet in the form of protein (Table 1). Whilst a high
protein requirement might have been expected for carnivorous fish species, such as plaice
(Pleuronectes platessa-50%; Cowey et al, 1972) or snakehead (Channa micropeltes-52%;
Wee and Tacon, 1982), the fact that a relatively high protein requirement was also observed
in the herbivorous grass carp (Ctenopharyngodon idella 41–43%; Dabrowski, 1977) suggests
that the requirement may in part be a function of the methodology used for its determination.
The use by different workers of different dietary protein sources, non-protein energy
substitutes, feeding regimes, fish age classes and methods for the determination of dietary
energy content and dietary requirement leaves little common ground for direct comparisons
to be made within or between fish speceis. For example, the high protein requirement
observed for grass carp fry (41–43%; Dabrowski, 1977) almost certainly arose from all
experimental fish being fed a restricted ration (fish fed only twice daily, and fixed on the
lowest recorded ad libitum feed take) and consequently fish fed the lower protein diets not
being able to consume sufficient feed to meet their dietary protein and energy requirements.
A critical review of the methods used for the estimation of dietary protein and amino acid
requirements in fish and crustacea has been made by Tacon and Cowey (1985) and Cowey
and Tacon (1983) respectively.

The high dietary protein requirement of fish and shrimp is generally attributed to their
carnivorous/omnivorous feeding habit, and their preferential use of protein over
carbohydrates as a dietary energy source (Cowey, 1975). In contrast to terrestrial farm
animals fish and shrimp are able to derive more metabolizable energy from the catabolism of
proteins than from carbohydrates.

2.3.3 Abiotic factors - temperature and salinity

The influence of water temperature on protein requirement and fish growth has been the
subject of numerous investigations. The early study of DeLong and co-workers with
fingerling Chinook salmon (O. tshawytscha) was said to show an increase in the dietary
protein requirement from 40% to 55% with an increase in water temperature from 8.3°C to
14.4°C (DeLong, et al., 1958). More recently, a similar rise in dietary protein requirement
was reported in fingerling Striped bass (Morone saxatilis) from 47% to 55% with an increase
in water temperature from 20.5°C to 24.5°C (Millikin, 1983; Table 1). In contrast, fingerling
rainbow trout (Salmo gairdneri) showed no difference in growth at dietary protein levels of
35%, 40% and 45% at temperatures of 9°C, 12°C, 15°C and 18°C in one study (Slinger et al.,
1977) or in another study with temperatures of 9°C, 15°C and 18°C (Cho and Slinger, 1978).
Although distinct temperature effects were observed in terms of growth, the greater absolute
need for protein at the higher water temperatures was apparently satisfied through the
increased consumption of the lower protein diets. These latter studies are in line with the
hypothesis that an increase in water temperature (up to an optimum level) is accompanied by
an increased feed intake (Brett, et al., 1969; Choubert, et al., 1982), increased growth rate and
metabolic rate (Jobling, 1983) and a faster gastro-intestinal transit time (Fauconneau, et al.,
1983; Ross and Jauncey, 1981) under conditions where food supply is not limiting. The
weight of evidence is that increased water temperature does not lead to an increased protein
requirement. In both cases where such a requirement was claimed, the effect of water
temperature on dietary protein requirement was investigated by comparing the results
obtained in successive experiments at different water temperatures. In addition, the sub-
optimal growth and increased feed intake observed with fish fed the higher protein diets
suggests that the ad libitum feeding regime employed in fact led to a restricted feed intake.

TABLE 1. Dietary protein requirement of fish and shrimp (expressed as % of dry diet)

Dietary
Feeding Culture
protein Size range 1
regime 2 system
Species requirement Reference
FISH
Oreochromis
40 Fingerling 6%bw/d Indoor/tank Jauncey (1982)
mossambicus
Oreochromis Santiago et al.,
35 Fry 15%bw/d Indoor/tank
niloticus (1982)
De Silva &
O. niloticus 28–30 Fry/fing. 6%bw/d Indoor/tank
Perera (1985)
Wang, Takeuchi
O. niloticus 25 Fingerling 3.5%bw/d Indoor/tank & Watanabe
(1985)
Teshima,
O. niloticus 35 Fingerling 4%bw/d Indoor/tank Kanazawa &
Uchiyama (1985)
Wannigama,
Weerakoon &
O. niloticus 19–29 Juvenile 3%bw/d Outdoor/cage
Muthukumarama
(1985) a
O. niloticus/aureus 2– Viola & Zohar
30 Grower Outdoor/pond
hybrids 2.5%bw/d (1984) b
Oreochromis Toledo, Cisneros
30 Fingerling 6%bw/d Indoor/tank
aureus & Ortiz (1983)
O. aureus 36 Fingerling 8.8%bw/d Indoor/tank Davis &
 Stickney (1978)
Winfree &
O. aureus 56 Fry 20%bw/d Indoor/tank
Stickney (1981)
Winfree &
O. aureus 34 Fingerling 10%bw/d Indoor/tank
Stickney (1981)
Mazid et al.,
Tilapia zilli 35 Fingerling 5%bw/d Indoor/tank
(1979)
Teshima,
T. zilli 35–40 Fingerling 4%bw/d Indoor/tank Gonzalez &
Kanazawa (1978)
Cyprinus carpio 35 Grower 5%bw/d Indoor/tank Jauncey (1981)
Murai et al.,
C. carpio 34 Fingerling Ad.lib. Indoor/tank
(1985)
Ogino & Saito
C. carpio 38 Fingerling Ad.lib. Indoor/tank
(1970)
Ctenopharyngodon Dabrowski
41–43 Fry Fixed (?) Indoor/tank
idella (1977)
Papaparaskeva-
Mugil capito 24 Fingerling Ad.lib. Indoor/tank Papoutsoglou &
Alexis (1985)
Fixed (1–
Ictalurus punctatus 35 Grower Outdoor/cage Lovell (1972) c
4%bw/d)
Fixed (1– Prather & Lovell
I. punctatus 29–42 Grower Outdoor/pond
4%bw/d) (1973) d
Fixed (34–
I. punctatus 45 Grower Outdoor/pond Lovell (1975) e
45kg/ha/d)
Page & Andrews
I. punctatus 25 Grower Ad.lib. Indoor/tank
(1973)
Garling &
I. punctatus 36 Fingerling 3%bw/d Indoor/tank
Wilson (1976)
Fixed (3– Deyoe et al.,
I. punctatus 25 Juvenile Outdoor/pond
4%bw/d) (1968) f
Page & Andrews
I. punctatus 35 Juvenile/grow. 3%bw/d Indoor/tank
(1973)
Murai,
Alosa sapidissima 42.5 Fingerling Ad.lib. Outdoor/tank Fleetwood &
Andrews (1979)
Chuapoehuk &
Pangasius sutchi 25 Fry/fing. 10%bw/d Indoor/tank
Pthisoong (1985)
Chanos chanos 40 Fry 10%bw/d Indoor/tank Lim et al., (1979)
Channa Wee & Tacon
52 Grower 2%bw/d Indoor/tank
micropeltes (1982)
Kanazawa et al,
Fugu rubripes 50 Fingerling 10%bw/d Indoor/tank
(1980)
Chrysophrys 38.4 Fingerling/juv. Ad.lib. Indoor/tank Sabaut & Luquet
aurata (1973)
Morone saxatilis 47 Fingerling Ad.lib. Indoor/tank Millikin (1983)
M. saxatilis 55 Fingerling Ad.lib. Indoor/tank Millikin (1982) g
Nose & Arai
Anguilla japonica 44.5 Fingerling Ad.lib. Indoor/tank
(1973)
Micropterus Anderson et al.,
45.2 Fry/fing. Ad.lib. Indoor/tank
dolomieui (1981)
Micropterus Anderson et al.,
40–41 Fingerling Ad.lib. Indoor/tank
salmoides (1981)
Pleuronectes Cowey et al.,
50 Juvenile Ad.lib. Indoor/tank
platessa (1972)
Jobling &
Salvelinus alpinus 36–43.6 Juvenile/grow. Ad.lib. Indoor/tank
Wandsvik (1983)
Austreng &
Salmo gairdneri 42 Grower Fixed (?) Indoor/tank
Refstie (1979)
S. gairdneri 40 Fingerling/juv. Fixed Indoor/tank Satia (1974) h
Zeitoun et al.,
S. gairdneri 40–45 Fingerling/juv. Ad.lib. Indoor/tank
(1973) i
PRAWN
Macrobrachium 12- Millikin et al.,
40 PL 0.15g Indoor/tank
rosenbergii 5%bw/d (1980)
Boonyaratpalin
M. rosenbergii 15 PL 0.12g Fixed Outdoor/tank
& New (1982) j
Balazs & Ross
M. rosenbergii 35 PL 0.10g 5%bw/d Outdoor/tank
(1976) k
Stanley & Moore
M. rosenbergii 27 PL 1.90g 5%bw/d Outdoor/pond
(1983) l
SHRIMP
Bhaskar & Ali
Penaeus indicus 30–40 PL 1–42 day Fixed Indoor/tank
(1984) m
10–
P. indicus 43 PL 0.4–1.1g Indoor/tank Colvin (1976)
15%bw/d
Venkataramiah,
100-
Penaeus aztecus ≤40 PL 24–135mg Indoor/tank Lakshmi &
50%bw/d
Gunter (1975)
Zein-Eldin &
P. aztecus 43–51 PL 0.4–1.3g Fixed (?) Indoor/tank
Corliss (1976) n
Andrews, Sick &
Penaeus setiferus 28–32 Juveniles 4g 5%bw/d Indoor/tank
Baptist (1972)
Penaeus AQUACOP
50–55 Juv. 3–8g Fixed (?) Indoor/tank
merguiensis (1978) n
Sedgwick (1979)
P. merguiensis 34–42 PL 0.3g Fixed (?) Indoor/tank n

Bages & Sloane

Penaeus monodon 55 PL 2mg Fixed (?) Outdoor/tank
(1981) o
P. monodon 34 PL 5mg 100%bw/d Indoor/tank Khannapa (1977)
100-
P. monodon 40 PL 25mg-0.7g Indoor/tank Khannapa (1977)
10%bw/d
AQUACOP
P. monodon 40 Juv. 1–3g Fixed (?) Indoor/tank
(1977) n
P. monodon 45.8 PL 0.5–1g Fixed (?) Indoor/tank Lee (1971) n
Smith et al.,
Penaeus vannamei ≥36 Juv. 4–20g Fixed (?) Indoor/tank
(1985) n
Colvin & Brand
P. vannamei 30–35 PL 32mg-0.5g (?) Indoor/tank
(1977)
Penaeus Colvin & Brand
30–35 PL 45mg (?) Indoor/tank
stylirostris (1977)
Colvin & Brand
P. stylirostris 44 PL 5mg (?) Indoor/tank
(1977)
Penaeus Colvin & Brand
44 PL 5mg (?) Indoor/tank
californiensis (1977)
Colvin & Brand
P. californiensis ≤30 Juv. 1g+ (?) Indoor/tank
(1977)
Deshimaru &
Penaeus japonicus 52–57 PL 0.8g Ad.lib. Indoor/tank
Yone (1978)
Balazs, Ross &
P. japonicus >40 Juv. 1–2g Fixed (?) Indoor/tank
Brooks (1973) n
Deshimaru &
P. japonicus 54 PL 0.6–1g Ad.lib. Indoor/tank
Kuroki (1974)
Forster & Beard
Palaemon serratus 30–40 PL 0.1–0.2g Fixed (?) Indoor/tank
(1973) n

1
Fish size range: fry 0–0.5g, fingerling 0.5–10g, juvenile 10–50g, grower 50g and above.
2
Feeding regime: %bw/d - fixed feed intake expressed as a percentage of body weight per
day, or Ad libitum feeding two to four times daily.
a
No difference in protein requirement at three stocking densities of 400, 600 and 800 fish/m3,
using 5m3 cages.
b
200m2 earthen ponds, fish density of 2/m2, ponds also fertilized with poultry litter at a rate
of 5kg/pond/week.
c
Fish stocking density of 300/m3.
d e
/ Fish stocking density of 9880/hectare.
f
Plastic lined ponds, with fish stocking density of 3000–3700/hectare.
g
Increased dietary protein requirement reported for fingerling striped bass from 47 to 55%
with an increase in water temperature from 20.5 to 24.5°C.
h
Feed intake fixed within all groups to the lowest recorded Ad libitum feed intake observed.
i
Protein requirement said to increase from 40 to 45% with increasing salinity.
j
Outdoor concrete ponds, 5 animals/m2, infrequent water exchange, all animals fed at same
fixed rate based on highest recorded intake.
k
outddor fibreglass tanks, 17 animals/m2, high water exchange.
l
Animals housed within pens in earthen pond, 10 animals/m2.
m
All animals fed at fixed rate of 5mg feed/larvae/day (PL 1–10), 15mg feed/larvae/day (PL
11–50), and 20mg feed/larvae/day (PL 24–42).
n
All animals fed to excess once or twice daily.
o
Diet formulated to 55% crude protein, but actual level after diet processing was 45%.

Very few studies have been undertaken concerning the effect of salinity on protein
requirement. Experiments conducted with fingerling rainbow trout ( a euryhaline fish) are
reported to show an increase in the absolute dietary requirement for protein from 40% to 45%
with a salinity increase from 10 to 20 parts per thousand (Zeitoun, et al., 1973; Table 1).
However, no increase in dietary protein requirement was observed in a similar experiment
conducted with Coho salmon fingerlings (O. kisutch); Zeitoun et al., 1974). In view of the
speculative method for arriving at dietary requirement from the dose response curve (Zeitoun
et al., 1973), and the lack of information on the protein requirements of these fish species in
full strength sea water (35 parts per thousand), there are no firm data demonstrating that the
protein requirements of fish are elevated with increased salinity. There is no information on
the effect of salinity on the protein requirement of shrimp.

2.4 Amino acids

Although over 100 different amino acids have been isolated from biological materials, only
25 of these are commonly found in proteins. Individual amino acids are characterised by
having an acidic carboxy group (-COOH) and a basic nitrogenous group (generally an amino
group: -NH2). In view of the presence of both acidic and basic groups, amino acids are
amphoteric (ie. have both acidic and basic properties) and consequently act as buffers by
resisting changes in pH. The chemical structure of the more commonly occurring amino acids
is shown below:
2.5 Amino acid function
Amino acids occupy a central position in cellular metabolism since almost all biochemical
reactions are catalysed by enzymes composed of amino acid residues. Amino acids are
essential for carbohydrate and lipid metabolism, for the synthesis of tissue proteins and many
important compounds (ie. adrenalin, thyroxine, melanin, histamine, porphyrins -
haemoglobin, pyrimidines and purines - nucleic acids, choline, folic acid and nicotinic acid -
vitamins, taurine - bile salts etc), and as a metabolic source of energy or fuel.

2.6 Amino acid requirements

For nutritional purposes, amino acids may be divided into two groups; the essential amino
acids (EAA), and the non-essential amino acids (NEAA). EAA are those amino acids that
cannot be synthesized within the animal body or at a rate sufficient to meet the physiological
needs of the growing animal, and must therefore be supplied in a ready made form in the diet.
NEAA are those amino acids that can be synthesized in the body from a suitable carbon
source and amino groups from other amino acids or simple compounds such as diammonium
citrate, and consequently do not have to be supplied in a ready made form in the diet.

The dietary EAA for fish and shrimp are as follows:

Threonine Valine
Leucine Isoleucine
Methionine Tryptophan
Lysine Histidine
Arginine Phenylalanine

Although the NEAA are not dietary essential nutrients, they perform many essential functions
at the cellular or metabolic level. They are termed dietary non-essential nutrients only
because the body tissues can synthesize them on demand. In fact it is often quoted that the
NEAA are physiologically so essential that the body ensures an adequate supply by synthesis.
From a feed formulation viewpoint, it is important to know that the NEAA's cystine and
tyrosine can be synthesized within the body from the EAA's methionine and phenylalanine
respectively, and consequently the dietary requirement for these EAA is dependent on the
concentration of the corresponding NEAA within the diet.

2.6.1 Optimum dietary essential amino acid levels

(a) Dose response and carcass deposition method: The quantitative EAA requirements of fish
have traditionally been determined by feeding graded levels of each amino acid within an
amino acid test diet so as to elicit a dose response curve (for review see Ketola, 1982; Cowey
and Luquet, 1983; Wilson, 1985). Dietary requirement is then usually taken at ‘break point’
on the basis of the observed growth response. In addition to growth, several workers have
also used free amino acid levels within specific tissue pools (whole blood, blood plasma or
muscle; Kaushik, 1979), or the oxidation of radioactively labelled amino acids (administered
orally or by injection; Walton, Cowey and Adron, 1982) as the criterion for estimating dietary
requirement. Within the amino acid test diets used the protein component is supplied almost
entirely in the form of crystalline amino acids or in combination with selected ‘whole’ protein
sources (commonly either casein, gelatin, zein, gluten or fish meal); the amino acid profile of
the total protein component of the diet being controlled so as to simulate the amino acid
profile of a specific reference protein.

In contrast to the above standard method where fish are fed graded levels of crystalline amino
acids, Ogino (1980a) determined the quantitative EAA requirement of fish simultaneously on
the basis of the daily deposition of individual amino acids within the fish carcass. In the
Ogino method fish are fed a diet containing a ‘whole’ protein source of high biological value,
and the dietary EAA requirement computed on the basis of the observed daily EAA tissue
deposition value.

Table 2 summarises the known quantitative EAA requirements of fish studied to date using
the above mentioned techniques. Quantitative dietary requirements for all 10 EAAs have
been established for only five fish species (common carp C. carpio, rainbow trout S.
gairdneri, channel catfish I. punĎtatus, Japanese eel A. anguilla, and the Chinook salmon O.
tshawytscha). At present there is no quantitative information on the dietary EAA
requirements of shrimp; in the main this has been due to the poor growth observed with
shrimp fed synthetic amino acid test diets and the inherent problems of nutrient leaching due
to the extended feeding habits of shrimp.

Although numerous independent studies have recently been performed on the amino acid
requirements of rainbow trout, significant differences in requirement (g amino acid/100g
protein) exist within and between individual fish species (Table 2). For example, differences
of the order of 65%, 72% and 114% were observed between independent laboratories for the
lysine, arginine and methionine requirement of fingerling/juvenile rainbow trout. Similarly,
inter-species variations ranged from 22% for valine to as high as 122% for tryptophan. Whilst
one would have expected the quantitative EAA requirements of fish to decrease with age and
decreasing protein synthesis (growth), one may well question whether or not the observed
variations in requirement are real or merely an artifact of the method employed. In contrast to
the variations in requirement observed for the same fish species fed conventional amino acid
test diets, there was no significant difference in the EAA requirement of carp and trout on the
basis of the carcass deposition method of Ogino (1980a). However, the dietary requirements
observed are within the range reported for fish fed amino acid test diets (Table 2).

Compared with the conventional method of feeding graded levels of individual amino acids,
the carcass deposition method of Ogino (1980a) offers numerous advantages:

 Fish are fed rations in which the protein component is supplied in the form of a
‘whole’ protein of high biological value. Amino acid requirements can therefore be
ascertained in fish displaying optimal growth.
 The dietary requirement for all ten EAAs can be determined simultaneously in one
single experiment. Using conventional amino acid test diets up to 10 separate
experiments have to be performed, each experiment involving the use of up to six
dietary regimes employing varying dietary concentrations of the single EAA under
test.
 Quantitative EAA requirements can equally be established for first feeding fry and
brood-stock fish with no loss of precision.

Table 2. Quantitative essential amino acid (EAA) requirements of selected fish species.
Values are expressed in order as a percentage of the dietary protein and as a percentage of the
dry diet (the denominator being the percentage protein in the diet)

Simulated amino acid

(AA) profile of Feeding Initial body
Species protein source regime 1 weight (g) Arginine
Cyprinus carpio Casein:gelatin (38:12) Ad.lib. 4f/d 0.5–4.0 3.3(1.3/38.5)
3%bw/d,
Ictalurus punctatus Whole hen's egg 2–10 4.3(1.03/24)
3f/d
Oncorhynchus
Whole hen's egg Ad.lib. 3f/d 2–4 6.0(2.4/40)
tshawytscha
Oncorhynchus keta Fish body protein Ad.lib. 2f/d 1.1 -
O. keta Fish body protein Ad.lib. 2f/d 1.1 -
Oncorhynchus kisutch Whole hen's egg Ad.lib. 3f/d 2–4 6.0(2.4/40)
Anguilla japonica ? ? ? 3.9(1.7/42)
Salmo gairdneri Whole hen's egg ? 12–14 >4.0(1.4/35)
5.4–5.9(2.5–
S. gairdneri Whole hen's egg Fixed (?) 1–2
2.8/47)
4.5%bw/d,
S. gairdneri Fish meal 1.5–9 -
3f/d
S. gairdneri Zein:fish meal (1:1) Ad.lib. 4f/d 20–30 3.43(1.2/35)
2%bw/d,
S. gairdneri Casein:gelatin (3:2) 27 -
3f/d
2–5%bw/d, 3.5–4.0(1.6–
S. gairdneri White cod muscle 5–14
4f/d 1.8/45)
1.5%bw/d,
Dicentrachus labrax Fish meal composite 35 -
2f/d
Oreochromis 4%bw/d,
Fish meal composite 1.7 <4.0(1.59/40)
mossambicus 3f/d
C. carpio Calculated on the basis of tissue deposition of 3.8(1.52/40)
S. gairdneri EAA, with fish fed a whole protein source of high 3.5(1.4/40)
biological value having a protein digestibility of
80%, and a feeding rate of 3% bw/d for both
species (carp 62–74g, 20–25°C; trout 68–127g,
15–18°C)

Species Histidine Isoleucine Leucine Lysine Methionine2 Methionine3

2.1(0.8/38.5 2.5(0.9/38.5 3.3(1.3/38.5 5.7(2.2/38.5 2.1(0.8/38.5) 3.1(1.2/38.5
C. carpio a
) ) ) ) )
1.54(0.37/24 2.58(0.62/24 3.5(0.84/24 1.34(0.32/24) 2.34(0.56/24
I. punctatus 5.1(1.5/30) b
) ) ) )
O.
1.8(0.7/40) 2.2(0.9/41) 3.9(1.6/41) 5.0(2.0/40) 1.5(0.6/40) c -
tshawytscha
O. keta 1.6(0.7/40) - - 4.8(1.9/40) - -
O. keta - - - - - -
O. kisutch 1.7(0.7/40) - - - - -
A. japonica 1.9(0.8/42) 3.6(1.5/42) 4.8(2.0/42) 4.8(2.0/42) 2.1(0.9/42) d 2.9(1.2/42)
S. gairdneri - - - 3.7(1.3/35) - -
S. gairdneri - - - 6.1(2.9/47) - -
1.57–
S. gairdneri - - - - 2.14(0.55– -
0.75/35) e
S. gairdneri - - - - - -
1–2(0.5–
S. gairdneri - - - - 1.0(0.5/50) f
1/50)
4.3(1.95/45
S. gairdneri - - - - -
)
D. labrax - - - - 2.0(1.0/50) h -
O. - - - 4.1(1.62/40 <1.33(0.53/40 -
mossambicu ) )g
s
4.1(1.64/40 5.3(2.12/40
C. carpio 1.4(0.56/40) 2.3(0.92/40) 1.6(0.64/40) -
) )
4.4(1.76/40 5.3(2.12/40
S. gairdneri 1.6(0.64/40) 2.4(0.96/40) 1.8(0.72/40) -
) )

Phenylalanine Phenylalanine Referenc

Species 4 5 Threonine Tryptophan Valine
e
3.4(1.3/38.5) 3.9(1.5/38.5 Nose
C. carpio h 6.5(2.5/38.5) 0.8(0.3/38.5) 3.6(1.4/38.5)
) (1979)
2.96(0.71/24 NRC
I. punctatus 2.0(0.5/24) i 5.0(1.2/24) 2.2(0.53/24) 0.5(0.12/24)
) (1983)
O. NRC
4.1(1.7/41) j - 2.2(0.9/40) 0.5(0.2/40) 3.2(1.3/40)
tshawytscha (1983)
Akiyama
O. keta - - 3.0(1.2/40) - - et al,
(1985)
Akiyama
0.73(0.29/40
O. keta - - - - et al,
)
(1985a)
Klein &
O. kisutch - - - 0.5(0.2/40) - Halver
(1970)
Nose
A. japonica 2.9(1.2/42) k 5.2(2.2/42) 3.6(1.5/42) 1.0(0.4/42) 3.6(1.5/42)
(1979)
Kim et
S. gairdneri - - - - - al.,
(1983)
Ketola
S. gairdneri - - - - -
(1983)
Rumsey
S. gairdneri - - - - - et. al.
(1983)
Kaushik
S. gairdneri - - - - -
(1979)
Walton
S. gairdneri - - - - - et al,
(1982)
Walton
0.45(0.25/55
S. gairdneri - - - - et al,
)
(1984)
Thebault
D. labrax - - - - - et al.,
(1985)
O. - - - - - Jackson
mossambicu &
s Capper
(1982)
Ogino
C. carpio 2.9(1.16/40) - 3.3(1.32/40) 0.6(0.24/40) 2.9(1.16/40)
(1980a)
Ogino
S. gairdneri 3.1(1.24/40) - 3.4(1.36/40) 0.5(0.2/40) 3.1(1.24/40)
(1980a)

1
Feeding regime: indicates feeding level and number of feedings per day.
2
In the presence of dietary cystine
a 2%;
b 0.24%;
c 1%;
d 1%;
e 0.3%;
f 2%;
g 0.74%;
h 1%
3
In the absence of dietary cystine.
4
In the presence of dietary tyrosine
h 1%;
i 1%;
j 0.4%;
k 2%
5
In the absence of dietary tyrosine.

(b) Carcass analysis method: Interestingly, recalculation of the data obtained by Ogino
(1980a) shows that there is no difference between the relative proportions of individual EAAs
required in the diet and the relative proportions of the same 10 EAAs present within the fish
carcass (Tacon and Cowey, 1985). A similar relationship has also been seen in the growing
pig and chick (Boorman, 1980), and to a lesser extent within the four fish species for which
EAA requirements have been determined using amino acid test diets (Fig. 3). Similarly,
Wilson and Poe (1985) obtained a regression coefficient of 0.96 when the EAA requirement
pattern for the channel catfish was regressed against the whole body EAA pattern found in a
30g channel catfish. Since the amino acid composition of fish body tissue does not differ
greatly (if at all) between individual fish species (Njaa and Utne, 1982; Wilson and Cowey,
1985), it follows, therefore, that the pattern of requirement for different species will also be
similar. Although not proven, it is not unreasonable to suppose that a similar relationship also
exists for shrimps and freshwater prawns. For comparative purposes Table 3 presents the
dietary EAA requirement pattern for fish, as determined by Ogino (1980a), together with the
carcass EAA pattern of whole fish body tissue, Penaeus japonicus
Figure 3 Relationship between pattern of EAA requirements found by feeding experiments
using amino acid test diets with carp (•), Japanese eel (■), channel catfish (□) and chinook
salmon (o) and the pattern of the same amino acids in fish carcass. The level of each amino
acid is represented as a percentage of the sum of all 10 EAA's in each pattern. The line
represents coincidence of requirement and tissue patterns. larvae and juveniles, Penaeus
paulensis juveniles, short-necked clam tissue (Venerupis philippinarum; regarded as an
excellent and ideal natural food for marine shrimp), and the tail muscle of Macrobrachium
rosenbergii. On the basis of the amino acid profiles presented it would appear that shrimp
have a higher dietary requirement for arginine, tryptophan and tyrosine, and a lower dietary
requirement for valine, threonine and lysine than fish.

Table 3. Mean fish dietary EAA requirement pattern (%) and EAA pattern in body tissue of
whole fish, short-necked clam, marine shrimp, and the freshwater prawn.

Fish Whole Short- P. P. P. M. rosenbergii

requireme body necked japonicus japonicus Paulensis tail muscle
nt (Ogino, fish clam larvae juveniles juveniles (Farmanfarmi
EAA 1980a) tissue tissue (Teshima, (Deshimar (unpublishe an & Lauterio,
 (Wilso (Deshimar Kanazaw u & d data) 1980)
n& u et al, a& Shigeno,
Cowey 1985) Yamashit 1972)
, 1985) a, 1986)
Threonine 10.6 9.2 9.6 5.9 8.2 6.7 7.5
Valine 9.5 9.5 8.5 8.8 8.3 13.6 7.3
Methionine 5.4 5.5 5.4 5.7 5.4 7.0 6.5
Isoleucine 7.5 8.0 6.8 9.1 8.6 6.9 7.4
Leucine 13.5 14.6 14.0 12.1 15.0 12.6 14.8
Phenylalani
9.5 8.3 7.7 8.6 9.0 9.2 7.3
ne
Lysine 16.8 16.9 14.7 13.1 15.8 15.4 17.1
Histidine 4.8 5.2 4.4 4.5 4.5 4.4 4.5
Arginine 11.6 12.3 15.5 14.1 15.2 14.3 20.6
Tryptophan 1.7 1.7 2.7 6.3 NA NA NA
Cystine * 2.7 2.0 2.7 2.4 2.1 3.0 NA
Tyrosine * 6.5 6.6 7.8 9.2 7.8 6.7 6.6

NA - data not available (not analysed).

* - Non-essential amino acids.All values are expressed as a percentage of total EAA plus
cystine and tyrosine.

In the absence of firm quantitative information on the dietary EAA requirements of shrimp
and the majority of farmed fish species, dietary requirement can initially be computed on the
basis of the carcass EAA pattern present within 35% of the known dietary protein
requirements of the said species; on a general basis EAAs (including the NEAAs cystine and
tyrosine) constitute about 35% of the total dietary protein required by fish (Table 2). Thus if a
shrimp or fish is known to have a dietary protein requirement of 45%, then dietary EAA
requirement would be computed on a carcass EAA pattern of 35% of the dietary protein
level. For example, if the carcass EAA pattern for lysine is 16.9% of the total EAA plus
cystine and tyrosine present, then dietary requirement level for lysine would be

or 2.66% of the dry diet (ie. 45% protein fish ration).

As a guide line Table 4 presents the calculated dietary EAA requirements of fish and shrimp
at varying dietary protein levels based on the mean carcass EAA pattern of whole fish tissue
and short-necked clam tissue respectively (short-necked clam tissue is used here in the
absence of a mean carcass EAA pattern for shrimp).

Table 4. Calculated dietary EAA requirements of fish and shrimp at varying dietary protein
levels (values are expressed as a percent of the dry diet)

Dietary protein level (%) Carcass

EAA
25 30 35 40 45 50 55 pattern
EAA (%)
 1
FISH
Arginine 1.07 1.29 1.51 1.72 1.94 2.15 2.37 12.3
Histidine 0.45 0.55 0.64 0.73 0.82 0.91 1.00 5.2
Isoleucine 0.70 0.84 0.98 1.12 1.26 1.40 1.54 8.0
Leucine 1.28 1.53 1.79 2.04 2.30 2.55 2.81 14.6
Lysine 1.49 1.77 2.07 2.37 2.66 2.96 3.25 16.9
Methionine 0.48 0.58 0.67 0.77 0.87 0.96 1.06 5.5
Cystine * 0.17 0.21 0.24 0.28 0.31 0.35 0.38 2.0
Phenylalanine 0.73 0.87 1.02 1.16 1.31 1.45 1.60 8.3
Tyrosine * 0.58 0.69 0.81 0.92 1.04 1.15 1.27 6.6
Threonine 0.80 0.97 1.13 1.29 1.45 1.61 1.77 9.2
Tryptophan 0.15 0.18 0.21 0.24 0.27 0.30 0.33 1.7
Valine 0.83 1.00 1.16 1.33 1.50 1.66 1.83 9.5
2
SHRIMP
Arginine 1.36 1.63 1.90 2.17 2.44 2.71 2.98 15.5
Histidine 0.38 0.46 0.54 0.62 0.69 0.77 0.85 4.4
Isoleucine 0.59 0.71 0.83 0.95 1.07 1.19 1.31 6.8
Leucine 1.22 1.47 1.71 1.96 2.20 2.45 2.69 14.0
Lysine 1.29 1.54 1.80 2.06 2.31 2.57 2.83 14.7
Methionine 0.47 0.57 0.66 0.76 0.85 0.95 1.04 5.4
Cystine * 0.24 0.28 0.33 0.38 0.42 0.47 0.52 2.7
Phenylalanine 0.67 0.81 0.94 1.08 1.21 1.35 1.48 7.7
Tyrosine * 0.68 0.82 0.96 1.09 1.23 1.37 1.50 7.8
Threonine 0.84 1.01 1.18 1.34 1.51 1.68 1.85 9.6
Tryptophan 0.24 0.28 0.33 0.38 0.42 0.47 0.52 2.7
Valine 0.74 0.89 1.04 1.19 1.34 1.49 1.64 8.5

1
Carcass EAA pattern of whole fish tissue (Wilson & Cowey, 1985)
2
Carcass EAA pattern of short-necked clam (Deshimaru et al., 1985)
* Non-essential amino acids

2.6.2 Utilization of free amino acids

Fish or juvenile shrimp fed rations in which a significant proportion of the dietary protein is
supplied in the form of ‘free’ or crystalline amino acids generally display sub-optimal growth
and feed conversion efficiency compared with animals fed protein-bound amino acids or
‘whole’ proteins (Wilson et al., 1978; Robinson et al., 1981; Yamada et al., 1981; Walton et
al., 1982; Deshimaru, 1981; Deshimaru & Kuroki, 1974a, 1975).

In general, dietary free amino acids are more rapidly assimilated in fish than protein-bound
amino acids. Experiments with rainbow trout (Yamada et al., 1981), common carp (Plakas et
al., 1980) and tilapia (Oreochromis niloticus; Yamada et al., 1982) fed free amino acid test
diets showed that peak plasma amino acid concentrations occurred sooner (12–24h, 2–4h, 2h,
respectively) than with an equivalent casein-based diet (24–36h, 4h, 4h, respectively).
Furthermore, in carp. individual free amino acids appear to be absorbed at varying rates from
the gastro-intestinal tract, and consequently peak plasma concentrations of individual amino
acids do not occur simultaneously (Plakas et al., 1980). In juvenile shrimp the situation
appears to be the reverse. For example, Deshimaru (1981) showed that the assimilation rate
of dietary free arginine into muscle protein by Penaeus japonicus juveniles was extremely
low (assimilation less than 0.6%) compared to that of protein-bound arginine (assimilation
above 90%). However, although Deshimaru (1981) reported no beneficial effect on growth of
free amino acid supplemented diets with P. japonicus juveniles, recent studies have
demonstrated that the larvae of the same species is capable of utilizing amino acid
supplemented diets for growth (Teshima, Kanazawa & Yamashita, 1986).

For optimal protein synthesis to occur, it is essential that all amino acids (whether they be
derived from whole proteins or amino acid supplements) are presented simultaneously to the
tissue. If such an equilibrium is not achieved, then amino acid catabolism (breakdown)
ensues with consequent loss of growth and and feed efficiency. For those warm water fish
species which display a rapid uptake and assimilation of free amino acids, it is therefore
essential that either; (1) the release or absorption of free amino acids from the diet is reduced
so as to minimise the variations in absorption rate observed between free and protein-bound
amino acids (achieved by coating individual amino acids with casein, zein or nylon-protein
membranes; Murai et al., 1982; Teshima, Kanazawa & Yamashita, 1986); or (2) that the
frequency of feed presentation is increased from two or three feeds per day to up to 18 feeds
per day so as to minimise the variations observed in plasma amino acid concentration
(Yamada, Tanaka & Katayama, 1981).

2.6.3 Amino acid composition and protein quality

On the basis of the above discussions it is evident that the protein quality of a feed ingredient
is dependent upon the amino acid composition of the protein and the biological availability of
the amino acids present. In general, the closer the EAA pattern of the protein approximates to
the dietary EAA requirement of the species, the higher its nutritional value and utilization.
For example, Table 5 presents the ‘chemical score’ or potential protein value of some
commonly used feed proteins. Chemical scores of 100 indicate that the level of a particular
EAA within the feed protein is identical to the dietary EAA requirement level for fish (when
expressed as a percentage of the total EAAs plus cystine and tyrosine) as determined by
Ogino (1980a). The chemical score of the protein is taken to be the percentage of the EAA in
greatest deficit relative to the dietary requirement pattern. This method of assessing protein
quality is based on the concept that the nutritive value of a protein depends primarily on the
amount of the EAA in greatest deficit in that protein, compared to a reference protein (in this
case the reference protein is the dietary EAA requirements of fish as determined by Ogino.
1980a). It can be seen from Table 5 that compared to fish meal or fish muscle, which has a
well balanced EAA profile and high chemical score (c. 80), the majority of protein sources
presented have amino acid imbalances which render them unsuitable as a sole source of
dietary protein for fish within complete diets intended for intensive farming systems. The aim
of feed formulation is to mix proteins of various qualities to obtain the desired EAA pattern
of the fish or shrimp species in question (complete diet feeding).

However, the above relationship between protein quality and EAA pattern will only hold true
if the individual amino acids are equally biologically available to the animal. For example,
under certain conditions some of the amino acids may be unavailable because the proteins in
the diet are incompletely digested. Thus, for carnivorous fish and shrimp species, the
cellulose cell wall within plant protein sources may render the proteins present within the cell
inaccesible to the digestive enzymes. In other cases, digestion may be hindered by the
presence of enzyme inhibitors within the food protein; trypsin inhibitor within raw soybeans.
Although it is possible to inactivate these inhibitors by moderate heat processing, under
conditions of excessive heat treatment proteins become more resistant to digestion due to
peptide bond formation occurring between the side chains of lysine and dicarboxylic acid.
The free epsilon amino groups of lysine are particularly susceptible to heat damage, forming
addition compounds with non-protein compounds (ie. reducing sugars such as glucose)
present in the food stuff (Cockerell, Francis & Halliday, 1972). This reaction is known as the
Maillard reaction, and renders the lysine biologically unavailable. Substances other than
reducing sugars which are known to react with the free epsilon amino group of lysine include
gossypol; phenol based compound present in cottonseed meal. An estimate of the biological
availability of amino acids within feed proteins, and hence an indicator of protein quality, can
be made by chemically measuring the free or available lysine content of the feed protein
(Cowey, 1979).

Table 5. Chemical score and limiting essential amino acids of some commonly used feed
proteins 1
1st
limiting
Feedstuffs Source2 Thr Val Met Cys Ils Leu Phe Tyr Lys His Arg Trp
amino
acid
Chick pea 1 64* 89 63* 104 119 110 113 86 72 100 166 129 Met
Mung bean 1 59* 110 54* 48* 127 121 124 94 79 114 123 123 Cys
Cow pea 1 65* 103 61* 59* 116 116 116 100 75 127 134 129 Cys
Yellow lupin 2 66* 81 20* 126 117 125 85 94 64* 117 192 135 Met
Lima bean 2 84 110 57* 74 135 118 125 106 72 112 98 106 Met
Broad bean 3 77 103 30* 41* 115 118 98 118 77 98 160 118 Met
Haricot bean 1 80 103 43* 67* 120 121 118 83 92 127 104 129 Met
Safflower 2 68* 125 63* 141 111 99 101 100 43* 121 181 118 Lys
Crambe 2 98 121 67* 218 117 104 83 86 66* 104 111 200 Lys
Palm kernel 2 62* 113 94 133 95 89 72 78 41* 98 225 311 Lys
Cottonseed 2 65* 102 52* 118 92 94 122 89 52* 117 205 141 Met/Lys
Sunflower 2 65* 124 83 137 115 104 109 91 42* 119 159 165 Lys
Linseed 2 71 122 93 156 111 90 105 92 43* 100 174 182 Lys
Sesame 2 58* 98 109 148 91 105 86 114 33* 114 211 153 Lys
Coconut 4 65* 114 61* 96 115 112 95 92 37* 81 217 123 Lys
Groundnut 4 55* 99 39* 133 117 100 107 117 53* 100 196 141 Met
Rapeseed 4 93 118 83 70 113 116 94 77 74 131 112 159 Cys
Soybean 4 74 101 46* 130 128 115 105 97 76 106 123 176 Met
Potato protein
5 89 125 63* 96 128 120 112 149 74 73 73 118 Met
concentrate
Leaf protein
6 84 127 57* 56* 112 120 122 129 71 90 96 141 Cys
concentrate
Spirulina maxima 2 87 136 52* 30* 159 118 105 123 55* 75 111 165 Cys
Saccharomyces
4 93 116 63* 85 139 112 91 108 86 106 89 141 Met
cerevisiae
Torulopsis utilis 4 94 118 54* 81 144 98 137 117 84 104 86 118 Met
M. methylotrophus 7 97 134 89 59* 115 107 115 138 71 83 84 118 Cys
Whole hen's egg 8 77 125 100 130 132 109 97 98 78 92 96 135 Thr
Fish muscle 9 83 98 98 85 108 110 80 117 101 121 97 135 Phe
Fish meal (herring) 4 76 127 109 78 117 107 80 95 89 96 111 123 Thr
Fish meal (white) 4 81 106 104 93 121 109 81 94 90 94 116 129 Thr/Phe
Fish protein
2 83 110 118 63* 127 109 85 103 92 90 95 153 Cys
concentrate
Fish silage 10 98 122 ---72--- 101 129 120 94 98 121 108 59* Trp
Whole shrimp meal 2 83 97 109 85 112 106 95 105 86 73 134 106 His
Meat and bone meal 4 77 128 59* 89 109 113 88 60* 86 100 150 88 Met
Blood meal 4 69* 158 33* 52* 24* 162 124 69* 89 214 62* 123 Ils
Liver meal 2 76 135 72 89 105 121 109 106 71 98 105 153 Lys
Poultry by-product
4 76 125 81 141 132 123 80 60* 71 87 134 112 Tyr
meal
Hydrolysed feather
4 91 164 24* 289 131 124 78 86 33* 50* 147 76 Met
meal
Worm meal 11 107 99 106 52* 112 124 84 108 79 125 98 82 Cys
House fly larvae 12 75 103 72 52* 96 90 128 218 77 127 82 147 Cys

1
Scores based on comparison with the mean essential amino acid requirements of rainbow
trout andcarp (Ogino, 1980). Mean EAA requirement (expressed as % of total EAA) being:
threonine 10.6;valine 9.5; methionine 5.4; cystine 2.7; isoleucine 7.5; leucine 13.5;
phenylalanine 9.5;tyrosine 6.5; lysine 16.8; histidine 4.8; arginine 11.6; and tryptophan 1.7
2
Source: 1-Kay (1979); 2-Gohl (1980); 3-Bolton and Blair (1977); 4-National Research
Council(1983); 5-Tunnel AVEBE Starches Ltd., UK; 6-Cowey et al. (1971); 7-Unpublished
data; 8-Coweyand Sargent (1972); 9-Connell and Howgate (1959); 10-Jackson, Kerr and
Cowey (1984); 11-Tacon,Stafford and Edwards (1983); 12-Spinelli (1980)

* Limiting essential amino acids (present below 30% mean fish requirement)

2.7 Evaluation of protein quality

Apart from chemically measuring amino acids and their availability within feed proteins,
there are many biological methods of evaluating protein quality:

Specific growth rate (SGR) The rate of growth of an animal is a fairly sensitive index of
protein quality; under controlled conditions weight gain being proportional to the supply of
essential amino acids. Daily SGR can be calculated by using the formula:
Food conversion ratio (FCR) Defined as the grams of feed consumed per gram of body
weight gain.

* As fed basis ie. dry weight

** Wet or fresh weight gain

Food efficiency (FE) Defined as the grams of weight gained per gram of feed consumed.
Units of expression as above.

Protein efficiency ratio (PER) Defined as the grams of weight gained per gram of protein
consumed.

* With this method no allowance is made for

maintenance: ie. method assumes that all
protein is used for growth.

Apparent net protein utilization (Apparent NPU) Defined as the percentage of ingested
protein which is deposited as tissue protein.

where Pb is the total body protein at the end of the feeding trial, Pa is the total body protein at
the beginning of the feeding trial, and Pi is the amount of protein consumed over the feeding
trial. In this calculation no allowance is made for endogenous protein losses. In contrast to the
previous methods of evaluating protein quality, this method requires that a representative
sample of animals be sacrificed at the beginning and end of the feeding trial for carcass
protein analysis.

The main drawback of these methods of predicting diet or protein quality is that they have to
be performed under controlled experimental conditions in the absence of natural food
organisms. Consequently, these methods can only be used within intensive or clear water
culture systems.

2.8 Nonprotein nitrogenous constituents

Amino acids are important not only as building blocks of protein but as the primary
constituents or nitrogen precursors for many nonprotein nitrogencontaining compounds.
Table 6 lists some of the more biologically important nonprotein nitrogenous compounds that
originate from amino acids.

Table 6. Nonprotein nitrogenous constituents derived from amino acids in animals1

Amino acid
Nitrogenous compound Physiological function of compound
precursor
Glycine & aspartic Constituents of nucleotides and
Purines & pyrimidines2
acid nucleic acids
Energy storage as creatine phosphate
Creatine Glycine & arginine
in muscle
Bile acids (glycoholic & Bile acids, aid in fat digestion and
Glycine & cysteine
taurocholic acids) absorption
Thyroxine, epinephrine &
Tyrosine Hormones
norepinephrine
Ethanolamine & choline Serine Constituents of phospholipids
Histamine Histidine A vasodepressor
Serotonin Tryptophan Transmission of nerve impulses
Constituents of haemoglobin and
Porphyrins Glycine
cytochromes
Niacin Tryptophan Vitamin
Melanin Tyrosine Pigment of skin and eyes

1
Lloyd, McDonald & Crampton (1978)
2
Pyrimidine and purine have been suggested to be essential dietary nutrients fornewly
hatched fish larvae (Dabrowski & Kaushik, 1982) and Artemia salina (Hernandorena, 1983)
respectively.

2.9 Protein and amino acid pathology

2.9.1 Dietary Essential Amino Acid Deficiency

Although all fish examined to date display reduced growth when fed EAA deficient diets, the
following additional gross anatomical deficiency signs have been observed under
experimental conditions with juvenile fish fed synthetic rations deficient in one or more
EAAs:

Limiting EAA Fish Deficiency signs1

Lysine Salmo gairdneri Dorsal/caudal fin erosions (1,2); increased mortality (2)
Cyprinus carpio Increased mortality (3)
Methionine S. gairdneri Cataract(4,5)
Salmo salar Cataract (6)
Tryptophan S. gairdneri Scoliosis2 (7–10); lordosis2 (7,10); renal calcinosis (8);
cataract (7,9); caudal fin erosion; decreased carcass
 lipid content (9); elevated Ca, Mg, Na and K carcass
concentration (7)
Oncorhynchus Scoliosis (11)
nerka
Miscellaneous O. keta Scoliosis/ lordosis (12)
Increased mortality and incidence of lordosis observed
C. carpio with dietary defi- ciencies of leucine, isoleucine, lysine,
arginine and histidine (3)

1
1-Walton, Cowey and Adron (1984); 2-Ketola (1983); 3-Mazid et al. (1978);4-Walton,
Cowey and Adron (1982); 5-Poston et al. (1977); 6-Barash, Poston andRumsey (1982); 7-
Walton et al. (1984); 8-Kloppel and Post (1975); 9-Poston andRumsey (1983); 10-Shanks,
Gahimer and Halver (1962); 11-Halver and Shanks (1960);Akiyama et al. (1985a).
2
Curvature of the vertebral column

Under intensive farming conditions dietary EAA deficiencies may arise from one of four
possible routes:

 Poor feed formulation arising from the use of disproportionate amounts of feed
proteins with natural specific EAA deficiencies (Table 5).

Dietary imbalances may also arise from the presence of disproportionate levels of
specific amino acids; including leucine/isoleucine antagonisms, and to a lesser extent
arginine/lysine and cystine/methionine antagonisms. For example, blood meal is a
rich source of valine, leucine and histidine, but is a very poor source of methionine
and isoleucine. However, in view of the antagonistic effect of excess leucine on
isoleucine, animals fed high dietary levels of blood meal suffer from an isoleucine
deficiency caused by an excess of dietary leucine (Taylor, Cole and Lewis, 1977).
Although similar antagonisms have also been reported for cystine/ methionine (use of
hydrolysed feather meal; Ichhponani and Lodhi, 1976) and arginine/ lysine (Harper,
Benevenga and Wohlhueter, 1970) in terrestrial farm animals, they have not been
found to occur in fish fed synthetic amino acid diet combinations (Robinson, Wilson
and Poe, 1981).

 Dietary EAA deficiencies may arise from excessive heat treatment of feed proteins
during feed manufacture.
 Dietary EAA deficiencies may arise from the chemical treatment of feed proteins with
acids (silage production) or alkalies, due to the loss of free tryptophan and
lysine/cystine respectively (Kies, 1981).
 Dietary EAA deficiencies may arise from the leaching of free and protein bound
amino acids into the water. For example, Grabner, Wieser and Lackner (1981)
reported the loss, through leaching, of almost all the free and about one-third of the
free plus protein bound amino acids from frozen or freezedried zooplankton (Artemia
salina and Moina spp.) after a 10 minute water immersion period at 9°C. Considerable
losses of water-soluble amino acids have also been observed in carp during
mastication (Yamada and Yone, 1986). However, the problem of nutrient leaching of
water soluble materials is probably greatest for crustaceans due to their very slow
demersal feeding habit and necessity to masticate their food externally prior to
 ingestion (Farmanfarmaian, Lauterio and Ibe, 1982). For example, Bages and Sloane
(1981) reported a 28% loss of dietary protein during the preparation and rehydration
of a dry alginate-bound shrimp diet prior to feeding, and a total protein loss of 39–
47% after a six hour immersion period in seawater. In general nutrient losses are
greater in freshwater than in seawater (Balazs, Ross and Brooks, 1973). However,
problems of nutrient leaching can be minimised by using an appropriate feeding
regime (ie. regular rather than infrequent feeding; Sedgwick, 1979) and a suitable diet
binding or micro-encapsulation technique (Goldblatt, Conklin and Duane Brown,
1980; Jones et al., 1976).

2.9.1 Toxic non-essential amino acids

Nutritional pathologies may also arise from the ingestion of feed proteins containing toxic
amino acids. Commonly used feed proteins which are known to contain toxic amino acids
include alkali-treated soybean (toxic amino acid - lysinoalanine), the legume Leucaena
leucocephala or ‘ipil ipil’ (toxic amino acid - mimosine), and the faba bean Vicia faba (toxic
amino acid - dihydroxyphenylalanine).

GIZI PENTING - PROTEIN DAN ASAM AMINO

2.1 Protein

Protein adalah salah satu unsur terpenting dari semua sel hidup dan mewakili kelompok kimia
terbesar di tubuh hewan, kecuali air; Bangkai seluruh ikan mengandung rata-rata 75% air, protein
16%, lipid 6%, dan abu 3%. Protein adalah komponen penting dari inti sel dan protoplasma sel, dan
karenanya memperhitungkan sebagian besar jaringan otot, organ dalam, otak, saraf dan kulit.
2.1.1 Komposisi

Protein adalah senyawa organik yang sangat kompleks dengan berat molekul tinggi. Kesamaan
dengan karbohidrat dan lipid, mengandung karbon (C), hidrogen (H), dan oksigen (O), namun selain
itu juga mengandung sekitar 16% nitrogen (N: kisaran 12-19%), dan kadang-kadang fosfor (P) Dan
belerang (S).
2.1.2 Struktur

Protein berbeda dari makromolekul biologis lain yang penting secara biologis seperti karbohidrat
dan lipid dalam struktur dasarnya. Misalnya, berbeda dengan struktur dasar karbohidrat dan lipid,
yang sering terdiri dari unit pengulang yang identik atau sangat mirip (yaitu unit pengulangan
glukosa di dalam pati, glikogen dan selulosa), protein mungkin memiliki hingga 100 unit dasar yang
berbeda ( asam amino). Oleh karena itu, keragaman dan rentang senyawa yang lebih besar
dimungkinkan, tidak hanya untuk komposisi, tetapi juga pada bentuk protein.
2.1.3 Sifat kimia

Koloid di alam, protein bervariasi dalam kelarutannya dalam air, dari keratin yang tidak larut ke
albumin yang sangat mudah larut. Semua protein dapat 'didenaturasi' oleh panas, asam kuat, alkali,
alkohol, aseton, urea dan garam logam berat. Ketika protein didenaturasi, mereka kehilangan
struktur uniknya, dan karenanya memiliki sifat kimia, fisik dan biologis yang berbeda (misalnya,
inaktivasi enzim oleh panas).
2.1.4 Klasifikasi

Protein dapat dikelompokkan menjadi tiga kelompok utama sesuai dengan bentuk, kelarutan dan
komposisi kimianya:

Protein berserat: protein hewani yang tidak larut yang umumnya sangat tahan terhadap
kerusakan enzim pencernaan. Protein berserat ada sebagai rantai filamen memanjang. Contoh
protein berserat meliputi kolagen (protein utama jaringan ikat), elastin (hadir dalam jaringan elastis
seperti arteri dan tendon), dan keratin (ada pada rambut, kuku, wol dan kuku mamalia).

Protein globular: mencakup semua enzim, antigen dan protein hormon. Protein globular dapat
dibagi lagi menjadi albumin (protein larut dalam air, protein koagulable panas yang terjadi pada
telur, susu, darah dan banyak tanaman); Globulin (tidak larut atau sedikit larut dalam air, dan hadir
dalam telur, susu, dan darah, dan berfungsi sebagai cadangan protein utama dalam benih tanaman);
Dan histones (protein dasar dengan berat molekul rendah, larut dalam air, terjadi di inti sel yang
terkait dengan DNA asam deoksiribonukleat).

Protein konjugasi: ini adalah protein yang menghasilkan kelompok non-protein serta asam amino
pada hidrolisis. Contohnya termasuk phosphoprotein (kasein susu, phosvitin dari kuning telur),
glikoprotein (sekresi mukosa), lipoprotein (membran sel), kromoprotein (hemoglobin, hemokianin,
sitokrom, flavoprotein), dan nukleoprotein (kombinasi protein dengan asam nukleat yang ada di Inti
sel).

2.2 Fungsi protein

Fungsi protein dapat diringkas sebagai berikut:

Untuk memperbaiki jaringan yang aus atau terbuang (perbaikan jaringan dan perawatan) dan
untuk membangun kembali jaringan baru (seperti protein dan pertumbuhan baru).

Protein makanan dapat dikotomatisasi sebagai sumber energi, atau dapat berfungsi sebagai
substrat untuk pembentukan karbohidrat atau lipid jaringan.

Protein diet diperlukan di dalam tubuh hewan untuk pembentukan hormon, enzim dan berbagai
macam zat biologis penting lainnya seperti antibodi dan hemoglobin.

2.3 Kebutuhan protein

Studi tentang kebutuhan nutrisi makanan pada ikan dan udang hampir seluruhnya didasarkan pada
penelitian yang sebanding dengan yang dilakukan dengan hewan ternak darat. Oleh karena itu,
hampir semua informasi yang tersedia tentang persyaratan nutrisi makanan dari spesies akuakultur
berasal dari percobaan pemberian makan berdasarkan laboratorium; Hewan yang disimpan di
lingkungan yang terkendali dengan kepadatan tinggi dan tidak memiliki akses terhadap organisme
makanan alami.
2.3.1 Tingkat protein diet optimal

Berdasarkan teknik pemberian makan yang dipelopori dan dikembangkan untuk hewan darat
kebutuhan protein protein ikan pertama kali diteliti pada salmon Chinnok (Oncorhynchus
tshawytscha) oleh Delong, dkk, (1958). Ikan diberi makan makanan seimbang yang mengandung
kadar protein berkualitas tinggi (kasein: campuran gelatin yang dilengkapi dengan asam amino
kristal untuk mensimulasikan profil asam amino protein telur ayam jantan) selama periode 10
minggu dan tingkat protein yang diamati memberikan pertumbuhan optimal. Diambil sebagai
persyaratan (Gambar 2). Karena studi awal ini pendekatan yang digunakan oleh pekerja saat ini telah
berubah sangat sedikit jika sama sekali, dengan kemungkinan pengecualian penggunaannya oleh
beberapa peneliti retensi protein jaringan maksimal atau keseimbangan nitrogen dalam preferensi
untuk penambahan berat badan sebagai kriteria kebutuhan (Ogino, 1980 ). Kebutuhan protein diet
biasanya dinyatakan dalam persentase makanan tetap atau rasio protein terhadap energi diet

Ara. 2. Kurva respon dosis tipikal

Sampai saat ini lebih dari 30 spesies ikan dan udang telah diperiksa dengan cara ini dan hasilnya
menunjukkan persyaratan protein diet yang seragam secara seragam dalam kisaran 24-57%, atau
setara dengan 30-70% kandungan energi kotor dari makanan dalam bentuk Protein (Tabel 1).
Sementara kebutuhan protein tinggi mungkin telah diperkirakan untuk spesies ikan karnivora,
seperti plaice (Pleuronectes platessa-50%; Cowey et al, 1972) atau snakehead (Channa micropeltes-
52%; Wee and Tacon, 1982), fakta bahwa sebuah Kebutuhan protein yang relatif tinggi juga diamati
pada ikan mas herbivora (Ctenopharyngodon idella 41-43%; Dabrowski, 1977) mengemukakan
bahwa kebutuhan sebagian dapat merupakan fungsi dari metodologi yang digunakan untuk
penentuannya. Penggunaan oleh para pekerja yang berbeda dari sumber protein makanan yang
berbeda, pengganti energi non-protein, rezim makan, kelas umur ikan dan metode untuk penentuan
kandungan energi makanan dan kebutuhan makanan tidak banyak memberi kesamaan bagi
perbandingan langsung yang harus dibuat di dalam atau di antara speceis ikan. . Sebagai contoh,
kebutuhan protein tinggi yang diamati untuk ikan mas rumput (41-43%; Dabrowski, 1977) hampir
dipastikan berasal dari semua ikan percobaan yang diberi ransum terbatas (ikan diberi makan dua
kali sehari, dan dipelihara pada pakan ad libitum terendah yang tercatat Mengambil) dan akibatnya
ikan memberi makan makanan protein rendah karena tidak dapat mengkonsumsi cukup pakan
untuk memenuhi kebutuhan protein dan energi diet mereka. Kajian kritis terhadap metode yang
digunakan untuk memperkirakan kebutuhan protein dan asam amino pada ikan dan krustasea
dilakukan oleh Tacon dan Cowey (1985) dan Cowey and Tacon (1983).

Kebutuhan protein tinggi ikan dan udang umumnya dikaitkan dengan kebiasaan makan karnivora /
omnivora mereka, dan penggunaan protein khusus mereka melebihi karbohidrat sebagai sumber
energi makanan (Cowey, 1975). Berbeda dengan hewan ternak darat, ikan dan udang mampu
memperoleh lebih banyak energi yang dapat dimetabolisme dari katabolisme protein daripada
karbohidrat.

2.3.3 Faktor abiotik - suhu dan salinitas

Pengaruh suhu air pada kebutuhan protein dan pertumbuhan ikan telah menjadi subyek banyak
penyelidikan. Studi awal DeLong dan rekan kerja dengan salmon Chinook fingerling (O. tshawytscha)
dikatakan menunjukkan peningkatan kebutuhan protein makanan dari 40% menjadi 55% dengan
peningkatan suhu air dari 8,3 ° C sampai 14,4 ° C ( DeLong, dkk., 1958). Baru-baru ini, peningkatan
kebutuhan protein dalam makanan serupa dilaporkan pada fingerling Striped bass (Morone saxatilis)
dari 47% menjadi 55% dengan peningkatan suhu air dari 20,5 ° C menjadi 24,5 ° C (Millikin, 1983;
Tabel 1). Sebaliknya, ikan trout pelangi fingerling (Salmo gairdneri) tidak menunjukkan perbedaan
pertumbuhan pada tingkat protein makanan 35%, 40% dan 45% pada suhu 9 ° C, 12 ° C, 15 ° C dan
18 ° C dalam satu studi ( Slinger et al., 1977) atau dalam penelitian lain dengan suhu 9 ° C, 15 ° C dan
18 ° C (Cho dan Slinger, 1978). Meskipun efek suhu yang berbeda diamati dalam hal pertumbuhan,
kebutuhan protein yang lebih besar pada suhu air yang lebih tinggi tampaknya terpenuhi melalui
peningkatan konsumsi makanan protein rendah. Studi terakhir ini sejalan dengan hipotesis bahwa
peningkatan suhu air (sampai tingkat optimum) disertai dengan peningkatan asupan pakan (Brett, et
al., 1969; Choubert, et al., 1982), tingkat pertumbuhan meningkat Dan tingkat metabolisme (Jobling,
1983) dan waktu transit gastro-intestinal yang lebih cepat (Fauconneau, et al., 1983; Ross dan
Jauncey, 1981) dalam kondisi dimana persediaan makanan tidak terbatas. Bobot bukti adalah bahwa
kenaikan suhu air tidak menyebabkan peningkatan kebutuhan protein. Dalam kedua kasus di mana
persyaratan semacam itu diklaim, efek suhu air pada kebutuhan protein diet diselidiki dengan
membandingkan hasil yang diperoleh dalam eksperimen berturut-turut pada suhu air yang berbeda.
Selain itu, pertumbuhan suboptimal dan peningkatan asupan pakan yang diamati dengan ikan yang
diberi pakan protein tinggi menunjukkan bahwa rezim pemberian makan libitum yang digunakan
sebenarnya menyebabkan asupan pakan terbatas.

2.4 Asam amino

Meskipun lebih dari 100 asam amino yang berbeda telah diisolasi dari bahan biologis, hanya 25 di
antaranya yang umum ditemukan pada protein. Asam amino individu dicirikan dengan memiliki
gugus karboksi asam (-COOH) dan gugus nitrogen dasar (umumnya gugus amino: -NH2). Mengingat
adanya kelompok asam dan basa, asam amino adalah amfoter (yaitu memiliki sifat asam dan sifat
dasar) dan akibatnya bertindak sebagai penyangga dengan melawan perubahan pH. Struktur kimia
dari asam amino yang lebih sering terjadi ditunjukkan di bawah ini:

1354/5000
2,5 Fungsi asam amino

Asam amino menempati posisi sentral dalam metabolisme sel karena hampir semua reaksi
biokimia dikatalisis oleh enzim yang terdiri dari residu asam amino. Asam amino sangat
penting untuk metabolisme karbohidrat dan lipid, untuk sintesis protein jaringan dan banyak
senyawa penting (yaitu adrenalin, tiroksin, melanin, histamin, porfirin - hemoglobin,
pirimidin dan purin - asam nukleat, kolin, asam folat dan asam nikotinat - Vitamin, garam
taurin - empedu dll), dan sebagai sumber metabolisme energi atau bahan bakar.
2.6 Persyaratan asam amino

Untuk tujuan nutrisi, asam amino dapat dibagi menjadi dua kelompok; Asam amino esensial
(EAA), dan asam amino non-esensial (NEAA). EAA adalah asam amino yang tidak dapat
disintesis dalam tubuh hewan atau pada tingkat yang cukup untuk memenuhi kebutuhan
fisiologis hewan yang sedang tumbuh, dan karenanya harus disertakan dalam bentuk siap
pakai dalam makanan. NEAA adalah asam amino yang dapat disintesis dalam tubuh dari
sumber karbon dan gugus amino yang sesuai dari asam amino lain atau senyawa sederhana
seperti diammonium sitrat, dan akibatnya tidak harus disertakan dalam bentuk siap pakai
dalam makanan.

EAA untuk ikan dan udang adalah sebagai berikut:

Threonine Valine
Leucine Isoleucine
Metionin Tryptophan
Lisin Histidin
Arginine fenilalanin

Meskipun NEAA(non-essential amino acids) bukan nutrisi penting makanan, mereka melakukan
banyak fungsi penting pada tingkat seluler atau metabolik. Mereka disebut nutrisi non-esensial diet
hanya karena jaringan tubuh dapat mensintesisnya sesuai permintaan. Sebenarnya sering dikutip
bahwa NEAA secara fisiologis sangat penting sehingga tubuh memastikan pasokan yang cukup
dengan sintesis. Dari sudut pandang formulasi pakan, penting untuk diketahui bahwa sistin dan tirosin
NEAA dapat disintesis di dalam tubuh dari metana dan fenilalanin EAA masing-masing, dan
akibatnya kebutuhan diet untuk EAA ini bergantung pada konsentrasi NEAA yang sesuai di dalam
diet

2.6.1 Tingkat asam amino esensial diet yang optimal

(A) Cara uji dosis dan pengendapan bangkai: Kebutuhan EAA kuantitatif ikan secara tradisional telah
ditentukan dengan memberi makan kadar asam amino masing-masing dalam diet tes asam amino
sehingga menghasilkan kurva respons dosis (untuk tinjauan lihat Ketola, 1982; Cowey dan Luquet,
1983; Wilson, 1985). Kebutuhan diet biasanya diambil pada 'break point' berdasarkan respons
pertumbuhan yang diamati. Selain pertumbuhan, beberapa pekerja juga menggunakan kadar asam
amino bebas di dalam kolam jaringan tertentu (seluruh darah, plasma darah atau otot; Kaushik,
1979), atau oksidasi asam amino berlabel radioaktif (diberikan secara oral atau dengan suntikan;
Walton, Cowey Dan Adron, 1982) sebagai kriteria untuk memperkirakan kebutuhan diet. Dalam diet
tes asam amino, komponen protein dipasok hampir seluruhnya dalam bentuk asam amino kristal
atau dikombinasikan dengan sumber protein 'keseluruhan' yang dipilih (umumnya berupa kasein,
gelatin, zein, gluten atau tepung ikan); Profil asam amino dari komponen protein total dari diet yang
dikontrol sehingga dapat mensimulasikan profil asam amino dari protein referensi tertentu.

Berbeda dengan metode standar di atas dimana ikan diberi pakan kadar asam amino kristal, Ogino
(1980a) menentukan kebutuhan EAA kuantitatif ikan secara simultan berdasarkan deposisi harian
asam amino individu di dalam bangkai ikan. Dalam metode Ogino, ikan diberi makan makanan yang
mengandung sumber protein bernilai tinggi 'keseluruhan', dan persyaratan EAA diet dihitung
berdasarkan nilai deposisi jaringan EAA harian yang diamati.

Tabel 2 merangkum persyaratan EAA kuantitatif yang diketahui dari ikan yang dipelajari sampai
sekarang dengan menggunakan teknik yang disebutkan di atas. Persyaratan diet kuantitatif untuk
semua 10 EAAs telah ditetapkan hanya untuk lima spesies ikan (ikan mas umum C. carpio, trout
pelangi S. gairdneri, lele saluran I. punĎtatus, belut Jepang A. anguilla, dan salmon Chinook O.
tshawytscha). Saat ini tidak ada informasi kuantitatif mengenai persyaratan diet EAA udang; Pada
pokoknya ini disebabkan oleh pertumbuhan yang buruk yang diamati dengan diet asam amino asam
amino yang diawetkan dengan udang dan masalah inheren pencucian hara karena kebiasaan makan
udang yang diperpanjang.

Meskipun banyak penelitian independen baru-baru ini dilakukan pada persyaratan asam amino dari
trout pelangi, perbedaan kebutuhan yang signifikan (protein asam amino / 100g) ada di dalam dan di
antara spesies ikan individu (Tabel 2). Sebagai contoh, perbedaan urutan 65%, 72% dan 114%
diamati antara laboratorium independen untuk kebutuhan lisin, arginin dan metionin ikan trout
pelangi / pelari remaja. Demikian pula, variasi antar spesies berkisar antara 22% untuk valin hingga
122% untuk triptofan. Sementara seseorang memperkirakan kebutuhan EAA kuantitatif ikan
menurun seiring bertambahnya usia dan mengurangi sintesis protein (pertumbuhan), seseorang
mungkin mempertanyakan apakah variasi kebutuhan yang diamati tidak nyata atau hanya
merupakan artefak metode yang digunakan. Berbeda dengan variasi kebutuhan yang diamati untuk
jenis ikan yang sama yang diberi diet asam amino konvensional, tidak ada perbedaan yang signifikan
dalam kebutuhan EAA ikan mas dan ikan trout berdasarkan metode pengendapan karkas Ogino
(1980a). Namun, persyaratan diet yang diamati berada dalam kisaran yang dilaporkan untuk diet uji
asam amino ikan (Tabel 2).

Dibandingkan dengan metode konvensional untuk memberi makan kadar asam amino individu,
metode pengendapan karkas Ogino (1980a) menawarkan banyak keuntungan:

Ikan diberi makan ransum dimana komponen protein dipasok dalam bentuk protein 'utuh' dengan
nilai biologis tinggi. Oleh karena itu, persyaratan asam amino dapat dipastikan pada ikan yang
menunjukkan pertumbuhan optimal.

Kebutuhan diet untuk semua sepuluh EAAs dapat ditentukan secara bersamaan dalam satu
percobaan tunggal. Dengan menggunakan diet tes asam amino konvensional hingga 10 percobaan
terpisah harus dilakukan, setiap percobaan yang melibatkan penggunaan hingga enam rejim diet
menggunakan berbagai konsentrasi diet dari EAA tunggal yang diuji.

Persyaratan EAA kuantitatif dapat sama-sama ditetapkan untuk pakan pertama dan stok ikan
induk tanpa kehilangan presisi.

Tabel 2. Persyaratan asam amino esensial kuantitatif (EAA) untuk spesies ikan terpilih. Nilai
dinyatakan dalam urutan sebagai persentase dari protein makanan dan sebagai persentase dari diet
kering (penyebut menjadi persentase protein dalam makanan)