Review: Phosphorus in Fish Nutrition

Phosphorus in Fish Nutrition 

Edited 6/3/2005 

Shozo H. Sugiura 

Introduction 

Phosphorus (P) is an essential nutrient for plants and animals. In animals, P plays numerous roles in intermediary 

metabolism, including phosphorylation of numerous proteins (e.g., enzymes, hormones, signaling proteins) for their 

activation, generation of high-energy carriers (e.g., ATP, creatin phosphate), and maintaining blood acid-base 

balance in kidney. P is an essential component of genetic materials (e.g., DNA, RNA), membrane phospholipids, 

hydroxyapatite of skeletal tissues (bones and teeth), and erythrocyte 2,3-diphosphoglycerate for oxygen delibery to 

tissues. Despite its pleiotropic roles in life, biochemical and molecular mechanisms of P deficiency have not been 

well characterized even in mammals. This is probably due to the fact that dietary P deficiency is rare in normal 

human dietitics. 

P is also critical for plants; P is a nutrient limiting the primary production of most aquatic ecosystems 

(Hakason & Carlsson 1998; Tyrrell, 1999; Mainston & Parr 2002). Thus, P input to the ecosystems can directly 

contribute to eutrophication, causing algal bloom and hypoxia of natural waters. In extreme cases, the P pollution 

can alter or even destroy aquatic habitats and create azoic environment. In the context of rapidly increasing 

aquaculture production as well as environmental awareness around the world, environmental regulatory agencies are 

making stringent guidelines to limit the amount of P that aquaculture industry can discharge into public waters. 

These guidelines not only reduce P pollution, but also reduce aquaculture production (Carlberg & Olst 2001). 

The continuous increase of aquaculture production is important for better human nutrition and poverty alleviation 

around the world (Tacon, 2001; Desai 2004). Therefore, it is imperative to improve/ develop technologies that can 

reduce environmental cost of aquaculture. The main challenge facing aquaculture today is to sustain dramatic 

increas es in fish production while minimizing environmental damage. 

Retention of dietary P by fish used to be only about 20%, and most dietary P was discharged to the 

environment (Philips & Beveridge 1986; Wiesmann et al. 1988; Holby & Hall 1991; Ketola & Harland 1993). 

Currently, however, fish can ret ain about 30-40% of P in typical commercial feeds (Green et al. 2002a, 2002b), 

which is a considerable improvement over the past figures. But, the ultimate goal is 100% P retention (zero 

emission). In order to increase P retention by fish, the mechanism of dietary P absorption and requirement will 

need to be clari fied. The present review dealt only with the deficiency, requirement, and bioavailability of P in fish 

nutrition. Other subjects of P nutrition were either omitted or given only cursory treatments. I therefore suggest 

the following reviews on fish P nutrition as complementary readings: Nose & Arai (1979), Ogino (1980), Lall 

(1989), Lall (1991), NRC (1993), Cowey (1995), Davis & Gatlin (1996), and Cho & Bureau (2001). 

Part 1. Phosphorus Deficiency & Requirement 

Critical factor: Growth magnification 

Growth is critical to induce P-deficiency or to study P-requirement. This important principle, however, has 

sometimes been overlooked even among contemporary scientists. Roloff (1875) fed dogs with a diet low in Ca, 

and produced rickets. He noted that the development of rickets depends on the size of the breed, the rapidity of 

growth, and the degree of deficiency of Ca in the diet. E. Voit (1880) fed puppies a mixture of meat and lard with 

or without Ca. He noted that those fed the diet without Ca supplement reduced not only Ca but also P in the bones. 

The animals were well-nourished and developed rickets. Voit also noted that rickets developed in direct proportion 

to the growth of the animal. Gustav von Bunge (undated) pointed out the relationship between the ash content of 

milk and the time required to double the weight of newborn animals of various species. For example, the required 

time for doubling the weight in the following species, man, horse, cow, and dog is 180, 60, 47, and 9 days, 

respectively. The percentages of ash in the milk of these species in the order named are 0.22, 0.41, 0.80, and 1.31. 

From this, Bunge concluded that the more rapidly the suckling grows, the greater the needs of the organism for those 

food stuffs which serve for the building up of the tissues, namely, proteins and salts. Hart, McCollum & Fuller 

(1909) reported that pigs fed a ration very low in P made as large gains up to 75-100 pounds, when starting at the 

© 2000, 2005. Shozo H. Sugiura. All rights reserved. 

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weight of 40-50 pounds, as animals receiving the same ration but supplemented with calcium phosphate. After 

reaching this point loss of weight began, followed by collapse. Pigs on the low-P ration maintained P levels in soft 

tissues and organs constant and comparable to those of normally fed pigs; however, they drew P from the skeleton, 

but removed Ca and P in the proportion found in tricalcium phosphate. Gregersen (1911) found in rats that even 

with an abundant intake of P in assimilable form, no P is retained from a protein-free diet. Mellanby (1919) noted 

that rickets developed much more readily in the fast-growing dogs than in those growing slowly. So, he 

characterized rickets "a disease of rapid growth". McCollum et al. (1921) found that the addition of butter to a 

rachitogenic diet, which was low in P and high in Ca, increased the growth of rats and as a result produced more 

severe rickets. 

Day & McCollum (1939) fed weaned rats with a diet containing only 0.017% P but otherwise adequate 

for growth. These workers observed that P-restrict ed rats grew and maintained a fairly good appetite for 2-4 weeks, 

then the animals gradually became inactive and used legs as little as possible, and died in 7-9 weeks on the deficient 

diet. The authors said, "The most striking effect of the P deficiency was on calcium . . . the loss of calcium is so 

much greater than of phosphorus." They also reported spontaneous fractures, and progressive rarefaction of bones 

by X-ray examination. The lethargic condition of the animals may be related to the low ATP level associated with 

P deficiency (discussed in the section: Metabolic responses). Gillis et al. (1948) fed chicks a diet containing 0.03% 

P, but otherwise capable to support optimum growth. The chicks ate well for 3 or 4 days and made small initial 

gains in weight. After this, there was a rapid decline in appetite, a general weakness, reluctance to stand or use legs, 

and lying on their sides. All chicks died between 5th and 10th day on the diet. These researchers are mentioning 

that there is a latent period in P deficiency, during which the animal is apparently (at least externally) normal. In 

undernourished human subjects, Rudman et al. (1975) noted that the retention of P, K, Na and Cl virtually halted 

when N (amino acids) was withdrawn from the otherwise complete hyperalimentation fluid. At all levels of N 

intake, these five elements including N retained in the body at a fixed ratio. Withdrawal of P also halted the 

retention of the other elem ents. When N, K, or P was withdrawn from the fluid, infused glucose continued to be 

utilized completely; however, a larger portion of glucose was used for lipogenesis than during infusion of the 

complete formula. Nose & Arai (1979) reported that Japanes e eel required 0.27% Ca and 0.29% P in diet for 

optimum growth. The highest weight gain of the fish during the feeding period (lasted 6-10 weeks ) was about 75% 

(of the initial wt) in the Ca experiment and only 45% in the P experiment. When growth magnification is low, the 

dietary requirement of most nutrients may well be underestimated if fish growth is used as the response criterion, 

whereas it could be overestimated if the retention or tissue concentration of test nutrients is used as the response 

criteria. For example, if feeding duration is too short, the requirement estimate based on growth can be zero, while 

that based on retention will be infinity (no plateau). Certain duration of feeding that allows suffi cient 

multiplication of the initial body size will be necessary in estimating the dietary requirements. Also, when feed 

efficiency is low, the dietary requirement will be low. In the P study, the fish gained only 45% during the 10 

week-feeding period, suggesting that the basal diet used in the experiment was of very poor quality or the rearing 

method was inadequate. In many studies dealing with large fish, the growth magnification tends to be small, which 

encounters a problem similar to this (see Section: P requirement for Large fish). Hardy et al. (1993) fed juvenile 

rainbow trout for 8 weeks with a P-deficient diet, a P-adequate diet or the mixture of these two diets at various ratios. 

Fish fed the P-defi cient diet showed clinical P-defici ency signs, including anorexia, transient lethargy, reduced 

growth, and dark coloration in 5 weeks, while fish fed the mixture of the P-defi cient and P-adequate diets at a 9:1 

ratio showed these signs in 7 weeks. Subclinical P defici ency did not affect fish growth until after the body P store 

was reduced below a certain threshold level. Storebakken et al. (2000) could reduce both the fecal and metabolic 

P excretion of Atlantic salmon by replacing fish meal in the diet with soyprotein concentrate. Total P content of the 

soyprotein diet and the fish meal diet was 1.2% and 1.8%, respectively. The fish growth did not differ at the end of 

the 84-day feeding period; however, the fish fed soyprotein diet had markedly lower P and Ca contents as well as 

Ca/P ratio in the whole body. The fish (initial wt. ~0.2kg) only doubled their weight during the experiment. The 

results clearly indicate that the growth does not respond until after body P store is reduced below a certain threshold 

level. The risk is that the initial body P store (pool size) is variable depending on the diet history of fish. The 

growth reduction can be immediate if the diet is P deficient and the fish do not have enough P savings in body. 

What are the Response Criteria? 

McCay et al. (1927) wrote "In evaluating the effectiveness of the diets we have employed two criteria, the rate of 

growth and the rate of death." This is a rational position to establish nutrient requirements since both criteria are 

practically important. Other responses such as feed efficiency, economical efficiency, disease resistance, fish 

(fillet) quality, and environmental effects are also self-explanatory. However, one wonders upon what grounds 


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maximizing the bone strength, blood P levels, tissue accumulation (saturation), and/or enzyme levels could be 

justified as the basis to establish the dietary requirement. Animals could be simply adapting to the change of 

dietary intake levels by responding physiologically in order to compensate for the decreas ed intake, which may not 

indicate or predict the possibility of clinical deficiency. Certain biological responses may not be the sign of clinical 

deficiency, and therefore when physiological responses are used (instead of clinical signs) to establish dietary 

requirem ents, the use of such responses needs to be justified first. In pigs and chickens, it is well established that 

the requirements of P for maximum bone strength and bone-ash content are higher than the requirements for 

maximum weight gain (Sauveur & Perez 1987, NRC 1998). Ogino & Takeda (1976) reported that the dietary 

requirem ent of available P for the maximum growth of juvenile carp (initial body wt. ca. 4 g, final wt. 7-12 g) was 

0.6-0.7%, and that for the maximum bone mineralization was higher (~1.5%) than that for optimum growth. 

Bureau & Cho (1999) reported that increasing dietary P intake had no significant effect on growth and feed 

efficiency but significantly increas ed P contents in the whole carcass, vertebrae, plasma, and urine. Rodehutscord 

(1996) demonstrated in rainbow trout (fish wt, initial 53 g, final max.200 g) that P requirement for maximum gain 

(3.7 g/kg diet) was lower than that for maximum P deposition or bone calcification (5.6 g/kg diet). He determined 

P requirement (?) based on various (nine) response indicators, which were all different. Such differences may be 

due to different sensitivity of the response indicators and due to different concepts of the requirement (i.e., 

requirem ent vs. saturation). Dougall et al. (1996) studied Ca and P levels in scales, vertebra, dorsal fin and serum 

of striped bass. They noted that ash, Ca, and P in bones and scales were sensitive, while serum P was not. The 

authors took an average of the requirements determined based on different response variabl es and from different 

trials (fish size, feeding period, etc. were different). Skonberg et al. (1997) reported that fish growth and feed 

efficiency were unaffected by dietary P levels (0.23-1.16%P) in a 8-week feeding trial with juvenile rainbow trout; 

however, ash, P, and Ca levels in the fish skin (with scales) and whole body were highly responsive to dietary P 

levels. The authors also noted that the plasma P and Ca levels and intestinal alkaline phosphatase levels were quite 

insensitive, while plasma alkaline phosphatase and body lipid levels showed some responses to dietary P levels. 

Eya & Lovell (1997) reported that channel cat fish (fish wt. initial 61 g; final 569-634 g) fed five di fferent diets of 

varied available P contents (from 0.2 to 0.6%) did not show any significant differences in weight gain, feed 

conversion, and dressing percentage in a 140-day feeding trial in earthen ponds. The diet had feed conversions 

between 1.7 and 2.0. Serum P, bone ash and bone P increased linearly, while muscle fat and visceral fat decreased 

linearly as dietary P increased. The dietary available P requirements to maximize serum alkaline phosphatase 

activity and bone-breaking strength were 0.25 and 0.31%, respectively. These authors also suggested a possibility 

of feeding more P than the minimum requirement to reduce fish body fat if there is an economical benefit. 

Response Criteria: Growth & Mortality 

Fordyce (1791) reported that when his canary hens were fed seeds many of the birds died, but when they received 

the same seeds and a piece of old plaster they were in good health. Fordyce concluded that canaries requi re a 

calcareous supplement to the seed diet. His experiment with fish, however, showed that fish were independent of a 

source of bone-forming materials. The fish were not given any food for months, but they grew rapidly and were 

healthy. Later, McCollum suggested in his book that somebody secretly fed the fish. Fish biologists, however, 

may be more inclined to say that the fish absorbed minerals from wat er and fed on planktons and algae in the tank. 

Knauthe (1898) reported that carp increas ed both N-retention and weight gain when meat ash was added to rations 

of meat meal and corn meal or meat meal and rice meal. Knauthe also reported that when meat ash was 

withdrawn from a meat meal-rice meal diet, digestibility of protein by carp decreas ed from 91.2% (with meat ash) to 

89.6% in the first 5 days and to 83.2% in the next 5 days. Digestibilities of fat and carbohydrates decreased 

likewise. He also reported that the fish reduced appetite, reduced body protein, and reduced fat deposition and fat 

synthesis from carbohydrates. Knauthe formulated a diet for mature carp, which contained more carbohydrates 

(as corn) and less protein (as meat meal) than a diet for young fish. The author suggested fortifying the former 

with basic calcium phosphate, probably because increasing carbohydrates resulted in a decrease of both P and Ca 

contents in the diet (cited in Higure 1912). McCay et al. (1927) noted that brook trout (initial BW ca. 2 g) fed a 

diet containing casein, starch, cod liver oil and yeast for 12 weeks were very listless and markedly abnormal 

compared with those fed the same diet but supplemented with Osborne & Mendel salt mixture. The growth, 

mortality, and body ash content were, however, not different. In this case, the primary response of fish to the 

dietary treatment is the behavior or appearance. Sekine et al. (1929) reported the result of a 63-day feeding trial 

conducted in 1927. They noted that the ash content of rainbow trout fry (initial wt. 0.18 g) was higher when the 

fish were fed a silkworm pupae-based diet supplemented with Osborne's salt mixture than when the fish were fed the 

same diet but without the salt mixture. Sekine & Kakizaki (1929) reported the results of a study conducted in 


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1926-27 in which they fed salmon fry for 38 days with either cooked rice, shark meal, sardine meal, or raw sardine 

as a sole diet. One group of fish was starved during the same period. A group of fish fed raw sardine had the 

highest survival (97%) and growth. The fish fed rice did not grow, and the survival rate was lower than the starved 

group. However, both starved fish and the fish fed on rice increased their body Ca content more than twice, while 

P content increased only slightly and Mg content decreased markedly during the period. The fish fed shark meal, 

sardine meal, or raw sardine increased the weight and the retention of Ca, P, Mg, N, and lipids. Sekine & Sato 

(1933) reported the results of a feeding trial conducted in 1930-31 with sockeye salmon (body wt., initial 0.17 g, 

final ca. 40 g; fed 391 days). The authors studied the supplemental effect of tricalcium phosphate (and Fe-citrate) 

using a diet containing fish meat (sic), silkworm pupae, rice bran, flour and a small amount of cod liver oil. The 

basal non-supplemented diet contained 23% protein, 55% carbohydrate, 13% lipids, 0.45% Ca, 0.85% P, and 

0.035% Fe (dry basis). Five grams of Ca 3(PO 4) 2 and 0.2 g of Fe-citrate were added to 700 g (dry wt) of the basal 

diet. The fish fed the P and Fe supplemented diet grew markedly better than those fed the basal diet; however, the 

survival rates (mortality) did not differ. The fish fed the P and Fe supplemented diet had higher percentages of ash, 

Ca, P, Fe and Mg in the body (dry basis) than the fish fed the basal diet, whereas the percent ages of protein and fat 

did not differ. Krockert (1938) fed brook trout with a diet containing 95% dried livestock blood, 4% dried 

potatoes, and 1% calcium phosphate. This diet was proven to support good growth of the fish, easily obtainable, 

and cheap. Increasing the amounts of potatoes (to 17.5%) and calcium phosphate (to 2.5%), and supplementing the 

diet with vitamins were suggested to increase the growth of the fish. 

Response criteria: Bones & Scales 

Gahn & Scheele (1770) found that the minerals in bones consist of calcium phosphate. Lavoisier (1790) wrote, 

"Phosphorus is found in almost all animal substances, and in some plants which give a kind of animal analysis." 

Bobba (1801) of Italy presented his theory on the cause of rickets. He thought, "by a derivation of the phosphat 

(sic) of lime from the bones to the joints (in rickets), symptoms of gout are produced, at the same time a 

mollification of the bones, which complication is named arthritis rachitica." He thought, "bad quality of the milk 

with which children are nourished is likely to be a frequent remote cause of the rickets." Johnson (1803) reported 

that chickens fed Ca phosphate had harder bones, and that Ca phosphate had also been fed profitably to children and 

pregnant women as a means of improving soft bones and healing fractures. Lawrence (1829-30) wrote, "In cases 

of rachitis, . . . we find less earthy matter and a greater proportion of animal substance than is natural. We find that 

the bones, in rickets, often admit of being cut with the knife." Brodhurst (1868) wrote a similar account. Also, 

May Mellanby (1918) made a similar comment on rachitic puppies, "the deficiency in calcium salts (in teeth) may 

result in the teeth being so soft that they can be cut with a scalpel." Guerin (1839), a French surgeon, fed some 

puppies on meat, and reported that they developed rickets; whereas the control animals, which were suckled, did not 

develop rickets. Chossat (1842, 1843) found that pigeons fed on wheat alone died in 10 months, during which 

period salts were gradually withdrawn from the bones, which thereby became fragile, and that this was prevented by 

giving a supplement of Ca carbonate. He mentioned that P in the wheat was not utilized because of defi ciency of 

Ca. Bibra (1844) published a book of 430 pages devoted to chemical analyses of bones of mammals, birds, 

reptiles and fishes. He showed deviations of ricketic, osteoporotic, and osteomalacic bones from the composition 

of normal bones in the proportion of organic to inorganic constituents. Bishop (1848) wrote, "During the period of 

the incubation of this disease (i.e., rickets) all the bones of the skeleton are more or less affected: they not only 

become soft and pliable, but their chemical and mechanical structure also undergoes a change." And further "They 

are lighter than natural, . . . being porous, soft, spongy, and compressible." Lehmann (1851) wrote, "In healthy 

human bones the phosphate of lime ranges from 48 to 59%; in softening of the bones it may sink to 30%. It is, 

however, singular that in almost all diseases of the bones, whether the results of osteoporosis, osteomalacia, or 

osteopsathyrosis, we find a diminution of the phosphate of lime (p. 413)." Anderson (1878) wrote, "rickets, which 

clearly shows, on chemical examination, is a defici ency disease of the inorganic matter . . . either the food is wanting 

in phosphate of lime, or there is a defect in its assimilation." And, "In rickets, bone becomes soft and pliable, 

yielding to any weight or strain put upon it, so that the lower limbs become bowed, the spine curved, and the 

cranium enlarged; the skeleton, from its imperfect construction, fails to fulfil the duties which properly belong to it. 

In rickets the inorganic deficiency is recogni zed, as productive of the disease, because the defici ency is obvious. 

The inorganic material bears a large proportion to the organic, and as the construction of bone is known, any great 

alteration in the relative proportion of organic and inorganic matter, is readily apparent; but in structures which show 

a small proportion of inorganic matter, deficiency of this may readily be overlooked . . . (p. 122)." Anderson 

(1878) presented numerous data of P contents (and major bases) in various tissues (tendon, skin, kidney, lung, brain, 

heart, aorta, and spleen) in various animal species (ox, pig, sheep, human) under normal and diseased states. The 


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author compared the differences in P contents and P/base balances among different organs and species. He also 

presented data of P contents in healthy urine and feces of humans on different diets. The author did not, however, 

directly study the effect of di fferent P intakes. According to Fernandes de Barros (undated), the ratio in which the 

carbonate of lime stands to the phosphate in the bones is 1:3.8 in the lion, 1:4.15 in the sheep, 1:8.4 in the hen, 1:3.9 

in the frog, and 1:1.7 in a fish. Fremy (undated) reported that scales of pike contained more ash (43% vs 34%), 

more calcium phosphate (43% vs 34%), and less organic metter (57% vs 66%) than those of carp. Weiske (1883) 

also reported that the scales of pike were lower in organic matter (58% vs 69%) and higher in ash (42% vs 31%) and 

P 2O 5 (18% vs 13%) than those of carp. He also reported that the vertebrae of carp and pike contained about 34% 

organic matter and 66% inorganic matter. The bone ash contained ca. 54% as CaO and 46% as P 2O 5 and trace 

amount of Mg. Heubner (1909) reported that rickets could be produced in dogs by feeding diets very low in P. 

He used egg-albumin as a source of protein. Hart et al. (1909) fed pigs for 3-4 months with diets of low-P content 

or with one of the following P supplements (precipitated Ca phosphate, bone ash, rock phosphate, phytin). They 

estimated the biological value of these P sources and approximate P requirement based on various responses such as 

weight gain, bone-breaking strength, bone ash content, bone density, bone wall thickness, bone diameter and Ca and 

P contents in various bones, blood, muscle, liver and other tissues of the body. They also estimated dietary P 

requirem ent based on P balance (intake minus excretions via feces and urine). Burnett (1906, 1910), Alway & 

Hadlock (1909), and Forbes (1909) conducted similar studies. Hess (1929) wrote, "No difference has been found 

in the potassium or sodium content of the bones in rickets. However, numerous investigators have reported an 

increas e in magnesium. Gassmann, for example, gives the figure of 0.1 per cent for normal bone and 0.53 per cent 

for rachitic bone, and states that there is a similar increase in the teeth (p. 147)." Hara (1930) studied N, ash, Ca 

and P contents in defatted-ground bones of 4yr-old mackerel, 2yr-old trout, sardine, pigs, rabbits, and dogs. The 

bones contained (in air-dry basis) 3.45-5.30%P in mackerel (n=6), 3.74-4.89%P in trout (n=2), and 2.61-4.71%P 

(n=6) in sardine. The author noted that bone Ca content tended to be higher in females than males, while bone P 

content was higher in males than females. Morgulis (1931) analyzed bone ash of various animal species for Ca, 

Mg, K, PO 4 and CO 2. He reported that the main difference between the chemical composition of the bone ash in 

marine fishes and in a variety of higher vertebrates was in the proportion of CaCO 3, which was only about one-half 

as large as in the latter. The author also mentioned that the PO 4/CO 3 ratio was variable, being markedly lowered by 

rickets and P-deficiency, and that the principal component of bone ash was Ca[{Ca 3(PO 4) 2} 6](OH) 2. Shimada & 

Kaneda (1937) reported that the bones of carp contained less ash, Ca and P than those of seabass, cod and seabream. 

Scales of carp was much lower in ash, Ca and P contents and higher in N content than those of sardine and saury. 

Takeuchi & Watanabe (1982) reported that ash, Ca, Mg, and P contents and the Ca/P ratio in vertebrae of carp 

(body wt. initial 13.2, end 8.6) did not change during 86 days of starvation (at 25°C) although body protein content 

decreased from 14.5 to 6.8% and body lipid content from 4.8 to 0.7% (wet basis) during this period. Percentages 

of ash and water (wet whole body) steadily increased from 3.2 to 4.2% and from 79 to 90%, respectively. These 

results suggest that starvation does not reduce bone minerals, while minerals in muscle including a certain amount of 

P will be lost. According to Lall (1991), the Ca/P ratio in scales and bones of various fish ranges from 1.5 to 2.1, 

and the ratio of Ca/P in the whole body ranges from 0.7 to 1.6. Hamada et al. (1995) studied bone ash of 15 fish 

species plus cattle, swine and fowl. They used both X-ray diffraction analysis to determine crystal structure of the 

bone ash and elemental analysis (13 elements analyzed) to determine the composition. The results showed that 

some fish had hydroxyapatite type bones, while others had tri-calcium type bones or intermediate of these two types. 

These authors suggested that since Ca of hydroxyapatite can be easily substituted by Mg and other elements, 

(Ca+Mg)/P ratio rather than Ca/P ratio may give accurate estimates for the theoretical value (i.e., Ca/P ratio in 

hydroxyapatite Ca 10(PO 4) 6(OH) 2, which is 1.67 or that in tri-calcium phosphate, which is 1.50). The (Ca+Mg)/P 

molar ratio that the author determined ranged 1.47-1.63. Cromwell (1989) stated that bone-breaking strength was 

more sensitive than bone ash content in pigs to determine relative bioavailability of P. Launer et al. (1978) used 

neutron activation analysis to determine P and Ca contents in fish samples, and reported that the contents of P in 

eviscerated channel catfish and its fat-free skeleton were highly variable depending on the sampling season. Bone 

P increased during wintering (non-feeding) period, but bone Ca decreas ed during the same period. For humans, 

non-invasive techniques are essential to measure bone density and to diagnose osteoporosis. Various techniques 

are used depending on the purpose and required accuracy: single- or dual-photon absorptiometry (SPA, DPA), 

single- or dual-energy x-ray absorptiometry (SXA, DXA), quantitative computed tomography (QCT), 

microdensitometry (MD), and quantitative ultrasound measurement (QUS) (Johnston et al. 1996). Schaafsma 

(1997) says that DXA is the method of choice to assess the bone mass, considering its accuracy and low radiation 

exposure. IOM (1997) used DXA to estimate bone mineral density and content, which was used to set dietary 

requirem ent of Ca for some age groups. This method was, however, not used to set the requirement of P (factorial 

method and serum Pi were used for P). Baeverfjord et al. (1998) studied bones of Atlantic salmon in relation to 


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fish growth by feeding either P-defici ent (0.35%P/diet) or P-suffi cient (0.9%P/diet) diets in freshwater or seawater 

environments. Fish grew properly for ca. 6 weeks on P-deficient diet, whereas whole body Ca and P levels 

declined immediately and soft, malform ed bones developed (e.g., wrinkly ribs, scoliosis). After 9 weeks, fish 

consuming P-deficient diet had whole body P and Ca levels 65% and 40% of the initial, respectively, and growth 

was almost halted. After 15 weeks of feeding with P-deficient diet, ash Ca, P, and Mg contents in bones and 

skin+scales decreased to 50% of the initial. However, plasma P and Ca concentrations did not differ between 

P-deficient and P-suffi cient fish. P-deficient fish had soft and bendable bones and opercula, but showed no 

fractures, which was considered as characteristic signs of P-deficiency that distinguish similar bone deformation but 

caused by vitamin C defici ency. Nine weeks of repletion feeding with P-suffici ent diet returned body P levels of 

P-deficient fish to normal; however, bone deformity was not completely reversible. These researchers used 

computerized tomography to measure bone density, and also Instron to compare structural strength of the bones. 

Response criterion: Blood P 

Whole blood of most animals contains 350-450 mg P per L as orthophosphate, most of which in the cells. The 

plasma inorganic P concentration is between 40 and 90 mg per L; much of the plasma P is ionized, but a small 

amount is complexed with proteins, lipids and carbohydrates (Bondi 1987). In humans, serum (or plasma) Pi 

ranges between 25 and 40 ppm, but it rises quite rapidly (peaks in ca.1 h) following the ingestion of food and returns 

to its pre-meal fasting level within 2-3 h. Also, after meals, insulin acts indirectly on cells to take up Pi (Anderson 

& Barrett 1994). The serum calcium concentration usually remains steady and under close endocrinological 

control by the hormones, parathyroid hormone and calcitonin. On the other hand, serum Pi is not so closely 

controlled, and concentrations vary widely. A fall in Ca concentration causes severe disruption of body function 

with tetany, paralysis, coma and death, while a fall of Pi imposes no immediate harm (Payne & Payne 1987). 

Iversen & Lenstrup (1919) and Howland & Kramer (1921) showed that rickets is associated with a 

definite chemical alteration of the blood, that the concentration of Pi is decreas ed, while the serum calcium being 

maintained approximately at the normal level. Kramer & Howland (1922) studied normal and rachitic rats, and 

noted that when the inorganic P of the serum is low it may be increased by (1) a few days of starvation, (2) by 

addition of inorganic P to the diet, (3) by addition of cod liver oil and (4) by exposure of the animals to UV 

radiations. Hess (1929) wrote, " . . . estimations of the inorganic phosphorus of the blood are of value. . . In 

general it holds true for experimental, as well as for infantile rickets, that the reduction in inorganic phosphorus 

parallels the intensity of the rachitic process (p. 70)." 

McCay (1931) determined total P, lipoid P, and acid-soluble P (organic and inorganic) in the whole blood, 

red cells, and plasma of pike, carp, bullhead (Ameiurus nebulosus), turtle, lampray-eel, congo-eel (Amphiuma 

tridactylum), and cows. The whole blood of carp and pike had ca. 4 times as much total P per unit volume as beef 

blood. P content in the blood plasma of fish was ca. 1-2 times higher than that of beef plasma, whereas P content 

in the red cells was 5-6 times higher in fish than in beef. Field, Elvehjem & Juday (1943) studied concentrations 

of P (inorganic and total) and other minerals in the blood of carp (and numerous organic constituents in the blood of 

both carp and brook trout). Phillips et al. (1957) determined a number of inorganic components including P in the 

blood of brown trout. They noted that inorganic P concentration in catfish, carp and trout blood was about twice 

the level of that in human blood. The authors warned that the nutritional status of fish and the active absorption of 

food could alter the P content of the blood. Phillips et al. (1961) reported that the blood of yearling brook trout 

contained 1270 mg P per L, of which 123 mg were contained in the acid-soluble fraction of the serum, and 44 mg in 

the acid-insoluble fraction. The total serum P content was therefore 167 mg/L. They found that the serum 32 P 

(and thus serum total 32 P and whole blood 32 P) increased rapidly through the first few hours after feeding 32 P in a 

Ca-free synthetic diet. The level started to decrease after 12 or 24 hours. This very rapid recovery of labeled 

dietary P in blood and other tissues of the fish convinced the authors to speculate that the direct passage of Pi 

through the wall of the stomach occurred (unknown in other species). They reported similar observations in 

several other experiments. The incorporation of 32 P into organic P in the serum began within 6 hours after feeding 

with gradual and regular rise. Fish excreted 16 times more 32 P when they were fed 4 mg of 32 P in diet compared 

with when they were fed the 1 mg level. In another experiment, they determined total P in the whole blood of 

fasting or fed (on practical diet) brook trout. The whole blood P level did not change by feeding (before vs aft er). 

The whole blood P of non-feeding fish was similar to that of fish fed a formulat ed diet, but slightly lower than those 

fed a meat diet. Fish held at 8°C had slightly higher whole blood P than those held at 11-16°C. Shimizu et al. 

(1963) reported seasonal changes of P, alkaline phosphatase and other components in the serum of yellowtail. 

Serum total P (ranged 378-712 ppm), Pi (58-137 ppm), Pi/total P ratio (0.11-0.34) and ALP activity (3.5-19.7 

nitrophenol unit/L) tended to be higher during summer than in winter. Under high stocking density, serum total P 


6

was lower and Pi/total P ratio was higher than when fish were stocked at normal density. McCartney (1971a) 

determined Pi, nucleic acid-P, phosphoprotein-P, lipid-P, barium-precipitated P in the erythrocytes of three trout 

species. McCartney (1971b) det ermined in brook trout the effect of dietary glucose, fructos e and galactose on 

plasma P levels at various fractions, including Pi, lipid-P, phosphoprotein-P, nucleic acid-P, fructose 6-phosphate and 

fructose 1,6-bisphosphate. Pi (153-171 ppm) and lipid-P (130-139 ppm) dominated the plasma total P (307-328 

ppm) regardless of the dietary treatments. Fish fed fructose, however, had higher levels of phosphoprotein-P and 

nucleic acid-P, and those fed glucose had higher levels of fructos e 6-phosphate and fructos e 1,6-bisphosphate than 

those fed the other nonsaccharides. In higher animals, administration of glucose or fructose is known to cause 

acute hypophosphatemia (Berner & Shike 1988, IOM 1997). Hammond & Hickman (1966) found that the 

plasma Pi of rainbow trout increased rapidly with strenuous exercise, but took 1-8 hours to return to the original 

level. Hille (1982) reviewed literatures on blood analyses of rainbow trout, and presented the normal ranges of 

various blood components and factors that might affect such measurements. He wrote: Cardiac sampling is the 

only suitable way since blood withdrawal from caudal vessels is subjected to contamination of tissue fluid. Plasma 

containing EDTA (as an anticoagulant) is unsuitable for the analyses of P, AP, and among others. The plasma 

concentration of phosphoprotein-P is about 0.25 ppm, which increases with estrogen during maturation. Serum 

phosphate does not depend on food phosphate supply (sic.). Compared with mammals, trout plasma contains large 

amounts of phospholipids, between 4610-8250 ppm. Hrubec & Smith (1999) reported that P values were higher 

in the serum than in plasma in all species tested (rainbow trout, hybrid striped bass, channel catfish, and hybrid 

tilapias). The authors suggested that the difference observed between serum and plasma most likely represents 

metabolic utilization of blood constituents while the blood was clotting. 

IOM (1997) used serum Pi concentration as the criteria to estimate dietary P requirement for humans. In 

turkey, however, Hurwitz et al. (1978) reported that plasma Pi increased linearly with absorbed P, while P retention, 

bone ash and weight gain all plateaued. Baeverfjord et al. (1998) did not find differences in plasma total P or Ca 

levels between P-depleted and P-suffici ent Atlantic salmon. Skonberg et al. (1997) concluded that blood plasma P 

levels were insensitive and fallacious as a response indicator of P status of fish because (1) blood P levels tend to 

reflect recent dietary intake more than the actual P status of the fish, (2) different methods measure di fferent 

fractions of P in the serum, plasma or whole blood (i.e., not only inorganic P but various acid-labile organic P 

compounds), and (3) non-nutritional factors, such as sampling time (aft er meal), handling of the samples, collection 

methods, temperature, salinity, and various stresses on fish when or before sampling, also have profound effects on 

the blood P level. The authors collected plasma from rainbow trout (body wt 1.9-5.3 g) that had been starved for 

1.5 days, and found little difference in the P concentration among fish fed diets of varied P levels. Vielma & Lall 

(1998b) reported a large difference in plasma P concentrations between samples collected 4 h after feeding and those 

collected 24 h aft er feeding. Thus, when estimating fish’s P status or adequacy of dietary P intake, the serum or 

plasma P, though sensitive, may not be reliable, unless the sampling procedure, especially feed intake of each fish 

and the postprandial sampling time, is well-controlled. Bureau & Cho (1999) reported the relationship between 

plasma P concentration and urinary P excretion. But, as these authors suggested, plasma Pi is highly variable over 

the whole day, and this may complicate the use of plasma Pi measurement to estimate urinary P output of fish (c.f. 

section “ Urinary P”). 

Response criterion: Urinary P 

Phosphorus was first discovered from urine in 1667 by Brandt who believed that human urine contained something 

that could convert silver into gold. Having been disappointed, he did not publish this discovery for years. Liebig 

(1842) wrote that the urine of carnivores is acidic and contains large amounts of alkaline bases united with uric, 

phosphoric, and sulfuric acids, and that these salts in blood are separated in the kidney. He believed that P in urine 

was derived from the "metamorphosed tissues" (p. 76). Liebig also wrote that the urine of gramnivora (e.g., 

horses) contains only traces of P, and a large amount of carbonates. He considered the difference in urinary P 

content between carnivora and hervivora as due to different rat es of tissue metamorphosis. Lehmann's book 

published in 1850 and translated into English in 1851 also reported a similar hypothesis, "The constant occurrence 

of phosphate of lime . . . obviously strengthen the opinion that this substance plays an important part in the 

metamorphosis of the animal tissues, and especially in the formation and in the subsequent changes of animal cells 

(p. 416)." Aubert (1852) found that while his normal daily excretion of phosphoric acid was 2.8g, it rose to 4.1g 

after he had swallowed 31g of phosphate of soda. Sick (1857) found that the ingestion of sodium P increased 

urinary P by more than the added amount, with a decrease of earth P and an increase of alkali P. Day (1860) wrote, 

"The quantity of phosphate of lime in the urine is dependent on the quantity of this substance occurring in the food, 

and on the demands of the organism for this salt. From the great demand on the part of the foetus for this 


7

substance, we commonly find that, during the latter months of pregnancy, the urine hardly contains more than traces 

of it, even when the diet has been sufficiently abundant (p. 128)." He also wrote, "The amount of the excreted 

phosphoric acid is . . . diminishing during prolonged abstinence, without, however, like the chloride of sodium, 

finally altogether disappearing (Schmidt observed that after prolonged abstinence, a cat excreted daily only 

one-third of the normal quantity of phosphoric acid. Mosler has made similar observations on man (p. 319)." 

Bischoff (1867) found that when dogs were fed lean beef P was excreted about 12-thirteenths in the urine, mostly 

combined with the alkalis, while the reminder left the body in the feces combined principally with Ca, Fe and Mg. 

On a diet of bread, a much larger amount of P appeared in the feces. Anderson's book published in 1878 is full of 

radical hypotheses (mostly incorrect, but entertaining) with few references. The basic theory that he called 

"capillary nutrition" was based on the observation that inorganic and nitrogenous materials were continuously 

excreted in urine, and on the assumption that various diseases were deficiency of available forms of mineral 

compounds in food. He wrote, "The diseases of nutrition, where the chemical composition of tissue undergoes 

alteration, are--- starvation, arising from general innutrition; fatty degeneration, dependent on organic innutrition 

(defici ency of albuminates in flesh); and the diseases which on the theory here advocated depend upon inorganic 

innutrition. These last are scurvy, rickets, scrofula, consumption, cancer, and leprosy (p. 94)." Anderson 

incorrectly concluded that urinary excretion of phosphate was due to matamorphosis of tissues (tissue renewal) and 

phosphorus used for brain and nerve, and thus the phosphorus excreted in urine was obligatory loss. From this, and 

his ballpark calculation of phosphorus balance, he suggested phosphorus deficiency was very likely (and thus causes 

"scurvy") (p. 110-111). He also wrote, "Many articles of food materially increase the quantity of phosphoric acid 

voided in the urine. An invalid, fed on milk alone, passes 5.5 grammes of phosphoric acid in his urine; beer, also, 

must have the effect of increasing the secretion of this acid, as phosphoric acid exists in appreciable quantities in 

beer. Phosphoric acid arising from such sources as this can take no part in nutrition; in order to arrive at anything 

like an accurate estimate of the quantity of phosphoric acid taking part in nutrition, these sources of error should be 

avoided (p. 63)." Apparently, he was aware that any excess of dietary P is excreted in urine and is useless in 

nutrition. Leder (1881) fed dogs with 500 or 1000g of meat of either raw or cooked, and found that the peak 

excretion of P and sulfur in urine occurred during the second hour, while the nitrogen peak did not occur until 4th 

hour. All had dropped to normal in 12 hours. In humans, North (1883) found that about equal quantities of P in 

foods were excret ed by the kidney and by the bowel. Gevaerts (1901) found that P excretion in the urine of white 

rats consuming a P-free diet was very much less in the amount than P present in the urine during starvation; and that 

on a ration of sucrose and edestin, or on sucrose and ovalbumin, there was much less P in the urine than on a ration 

of sucros e alone. Plimmer et al. (1909-1910) found that Pi in urine constitutes 90-100% of the whole. They 

wrote, "The quantity (of Pi) excreted (in urine) depends entirely on the intake of P 2O 5 . . . (conversely) the daily 

output (of the organic P 2O 5) in our subject was extremely irregular; . . . The organic P 2O 5 in the urine is therefore 

uninfluenced by diet and must originate in the body, i.e. it must be endogenous." (-- words in parentheses were 

added). Maurel (1901, 1904) estimated daily P requirement in normal diets by experimenting on himself and 

based solely on the urinary P excretion. Heubner (1909) produced rickets by feeding two young dogs a 

P-deficient diet (egg-white based diet) for 7 weeks. Three other dogs received a high-P ration containing casein 

and P-supplement, etc. The P content of the urine differed greatly, and corresponded to the amount of P in the diet. 

The P content in the feces of these dogs, however, was similar throughout the period and was unrelated to the food. 

Wolf & Oesterberg (1911) noted that feeding starving dogs with a small amount of protein reduced urinary P 

excretion to a very low level, while feeding starch and fat had little or no effect on P excretion. Denis (1912-13) 

collected urine from dogfish (elasmobranch) quantitatively for 24 h. The fish were tied by means of inch wide 

bandages to a board about 2 feet in length, which was placed in a large tank of running seawater and fastened in 

such a position that the cloaca was raised above the surface of the water. The urine was collected through the 

cannula tied to the urinary papilla. Alternatively, some fish were placed in a narrow trough without fastening the 

fish to a board. The author noted that pithing (destroying) the spinal cord up to about the level of the dorsal fin was 

helpful. The urine was analyzed for many components, and the average P concentration of 10 dogfish (fasting) was 

4520 ppm as P 2O 5 (= 1973 ppm as P). The urine was acidic, and when neutralized earthy phosphates precipitated. 

Denis (1913-14) analyzed urine of goosefish (teleost) collected from the bladders of 6 fish after death. The urine 

contained 440 ppm as P 2O 5 (= 192 ppm as P). Smith (1930, 1932) demonstrated that essentially all of the 

absorbed P, Mg, Ca, and sulfate which leave the body are excreted by the kidney alone. In humans (Grosser 1920) 

and dogs (Salvesen 1923), it has been shown that even when calcium is injected subcutaneously, it is excreted 

mainly by the bowel; whereas under similar conditions P is passed in the urine, and no increase being noted in the 

feces. Grafflin (1936) concluded that the glomerular kidney of sculpin as well as the aglomerular kidney of the 

goosefish and toadfish excretes Pi that is derived from some unidentifi ed precursor other than Pi in the plasma. 

Kaune & Hentschel (1987) reported that goldfish excrete P by regulating renal tubular reabsorption as well as renal 


8

tubular secretion (as in birds and reptiles). When sodium phosphate was i.v. infused, 65% of the infused P was 

excreted renally and 75% of it was eliminated by tubular secretion. Using flounder proximal tubule monolayer 

cultures, Gupta & Renfro (1991) has shown that renal Pi secretion is also dependent on a Na-gradient. Miller et 

al. (1964) in a balance trial showed a proportionate increase of urinary P excretion in baby pigs receiving P in 

amounts higher than 0.5% in the diet, which resulted in similar P retention by the pigs consuming diets containing a 

higher amount of P. In turkey, Hurwitz et al. (1978) also reported that when P absorption was below ca. 200 mg/d, 

urinary P was essentially zero, whereas once P absorption exceeded that level, urinary P excretion (mg/d) increased 

as the P absorption increased. The increase was, however, somewhat non-linear. In humans, it has been known 

that P homeostasis depends primarily on the mechanisms that govern renal excretion, which is reduced to essentially 

zero within 24 h of starting a P free diet (Moser et al. 1981, Dennis 1992). Sugiura (1998) estimated dietary P 

requirem ent for large rainbow trout based on non-fecal (urinary) excretion of P, which was determined by sampling 

tank water. Rodehutscord et al. (2000) reported that the non-fecal P excretion of rainbow trout was 3.7 mg 

(average) per kg BW daily when dietary P level was low, but was progressively increased at higher dietary P intakes. 

The amount of non-fecal P excretion was calculat ed as Amount of diet fed × Total P% in diet × Apparent P 

absorption% – P retained by fish. Bureau & Cho (1999) studied the relationship between plasma P and urinary P 

excretion in rainbow trout using tritiated PEG as a glomerular filtration marker and urinary spot sampling. They 

reported that the plasma Pi threshold concentration for urinary Pi excretion was about 86 mg/L, and implied that 

formulating diets with available P content that can produce plasma Pi close to this threshold concentration be sought 

in order to minimize soluble P excretion by fish, while maintaining optimal health and performance of the fish. 

These investigators also demonstrated that high dietary Pi intake causes trout to excrete excess Pi in kidney via 

tubular secretion as well as via reduced tubular reabsorption. 

Response criterion: Alkaline phosphatase 

Kay (1930) found that the blood serum level of the enzyme phosphatas e was abnormally high in ricketic infants. 

This measurement was described as a more accurate way to diagnose rickets than taking X-ray or measuring blood 

serum inorganic P levels. Pileggi et al. (1955) found that intestinal and fecal alkaline phosphatase (AP) activities 

increas e in response to decreas ed dietary P intake. Shimizu et al. (1963) studied seasonal changes of alkaline 

phosphatase and other components in the serum of yellowtail as discussed above. Miller et al. (1964) found P 

deficient baby pigs had markedly elevated serum AP activities. Kempson et al. (1979) measured various 

enzymes in various tissues in normal and P restricted rats. P restricted rats had significantly higher AP activity in 

renal brush border, intestine, liver, heart, and plasma than the control group. Boyd et al. (1983) determined relative 

P availability of high-moisture corn (25% moisture) in pigs based on plasma AP activity and the slope-ratio 

technique. The plasma samples were collected at d 14 of consuming experimental diets containing graded levels of 

KH 2PO 4 (standard) or high-moisture corn replacing dextrose in the basal diet. The P availabilities estimated based 

on plasma AP activity (43.8%) closely agreed with that estimated based on bone-breaking strength (41.3%). Koch 

et al. (1984) also measured serum AP activity in pigs fed low, medium or high P diets for 21 or 35 days. In these 

studies with terrestrial animals, increased AP activity is an indication of low dietary P intake or P deficiency. In 

contrast to this general observation in terrestrial animals, P-defi ciency in fish seems to be associated with low AP 

activity in plasma or serum. Eya & Lovell (1997) noted that serum AP activity of channel catfish (body wt. ca.600 

g) increas ed in a quadratic manner from 14.8 to 19.0 units/L as the dietary P level increased from 0.2 to 0.6%. Eya 

& Lovell (1998) also noted that serum AP activity of small channel catfish (body wt. ca.25 g) increased from 17.5 to 

23.9 in a quadratic manner with dietary P levels. Skonberg et al. (1997), however, noted that both plasma and 

intestinal AP activity in rainbow trout fed diets of various P levels were very variable, and not correlated to dietary P 

intake. The reported values of plasma AP ranged 31-223 units/L. Shearer & Hardy (1987), however, reported a 

slightly higher (insignificant) plasma AP activity in rainbow trout fed a P-defici ent diet (208 units/L) than those fed 

a P-adequate diet (168 units/L). Coloso et al. 2003) reported that intracellular phytase activity increased, but 

brushborder alkaline phosphat ase activity decreased in the intestine, pyloric caeca, and gills of trout (bw 115 g) fed 

low dietary P (0.1-0.6% total P in semi-purified diet) for 23days. According to Hille (1982), normal values of AP 

in plasma of rainbow trout from 9 independent studies were 126 (median value) or 98-261 (range) units/L. 

Response criterion: Reproduction 

A rapid increase of organic P in the gametes seems to necessitate an extra supply of P in the diet during maturation. 

However, since P content in the ovary is much less than that in the whole body of rainbow trout (3200 vs 4800 ppm, 


9

Shearer 1984), fish could require less P to increase ovary (during maturation) than to increase body weight (during 

growth). The dietary P requirement, therefore, can be lower for broodfish than for young growing fish. However, 

since maturing fish change their feed intake and various metabolic (hormonal) balances from the normal, it is 

difficult to estimate their dietary P requirement without performing an experiment. Generally, however, the P 

requirem ent for breeding animals or laying birds is similar or less compared with the requirement for young growing 

ones. In humans, both P requirement and recommendation for pregnant or lactating adult women are the same as 

those for the adult, but are much lower than those for the adolescent (IOM 1997). Watanabe et al. (1984a) 

reported that a fish meal based diet containing 2.2% P was deficient in available P content for broodstock fish of red 

seabream. Fish fed the diet for 7 months had lower fecundity, and produced eggs and larvae of much lower 

hatchability and higher abnormality than those fed P-forti fied diet. However, the mineral contents of tissues 

(vertebrae, liver) and eggs of the fish did not differ (Watanabe et al. 1984b). In another study of longer feeding 

duration (Watanabe et al. 1984c), broodstock fish of red seabream fed P-unforti fied diet performed similar to or 

better than those fed P-forti fi ed diet. Watanabe (1985, 1988) also reported that the brood fish of ayu, Plecoglossus 

altivelis, fed P-unforti fied diet had lower growth and fecundity than those fed P-forti fied diet, whereas chemical 

composition of the eggs (P, Ca, ash, lipids and proximate) did not differ. These experiments were not replicated. 

Response criterion: Resistance to infection 

Resistance to infection is a unique criterion in P nutrition. There may be some indirect effects of P-defici ency 

secondary to anorexia that reduces intakes of all essential nutrients, or to increased body fat or possible changes in 

phospholipids and fatty acid profiles that could affect membrane fluidity and metabolism of prostanoids. P 

deficiency has been suggested to have several indirect effects on immune functions via 1,25-(OH) 2D3/VDR 

-mediated pathways in extrarenal tissues (Brown, 1999; Omdahl, 2002) and via depression of leukocyte functions 

associated with decreased ATP content (Knochel, 2000). However, little is known regarding the specific effects of 

P deficiency on disease resistance and immune functions in any animal species. Eya & Lovell (1998) fed juvenile 

channel cat fish (initial body wt 2.1 g) for 10 weeks, and estimated the dietary P requirement based on maximum 

alkaline phosphatase activity, survival from Edwardsiella ictaluri challenge, and weight gain, which ranged from 

0.38 to 0.42%P in diet. The authors concluded that the dietary P level influences the resistance of cat fish to the 

pathogen, but the requirement for maximizing the growth is sufficient for maximizing the resistance to the pathogen. 

When the fish were challenged with the pathogen, however, the fish were approximately 10 times different in size 

(2.4 g in P-deficient fish and 23.1 g in P-adequate fish). If the difference in mortality was simply due to the 

difference in fish size rather than to their P-status, the conclusion may be untenable even though the difference in 

size was caused by P-deficiency. Also, the concentration of ascorbic acid in diets might be different due to the use 

of acidic salts (NaH 2PO 4) that can reduce dietary pH, which in turn increases the stability of ascorbic acid in diets. 

Jokinen et al. (2003) reported that plasma IgM concentration of whitefish fed P-deficient diets decreased 

significantly compared with those fed P-siffi cient diets. Plasma lysozyme activity and the antibody response to 

bovine gamma globulin did not differ between P-deficient and P-sifficient fish, while the growth of P-defici ent fish 

was markedly lower. They concluded that dietary P-deficiency has only minor effects on immune functions of 

whitefish, and that the dietary P level that can support normal growth of the fish is sufficient to elicit normal immune 

functions. 

Biochemical / Metabolic responses to P-deficiency 

In 1821, Francois Magendie injected a mixture of P and oil into the circulation of a dog. Soon afterward the 

animal exhaled white fum es from its nose. Magendie explained that so long as the phosphorated oil was in contact 

with the blood, no reaction occurred, but as soon as it passed through the surface membrane of the lungs and came 

into contact with the air a combustion took place. It seems that Magendie was aware of the role of P as a key 

element in energy metabolism and lipid (beta) oxidation when little was known about P metabolism. Liebig (1843) 

wrote, "The production of fat is always a consequence of a deficit supply of oxygen, for oxygen is absolutely 

indispensable for the dissipation of the excess of carbon in the food (p. 85)." Liebig did not relate this to 

P-deficiency; however, because P-defi ciency causes cellular hypoxia it is natural to find increased fat-deposition in 

P-deficient animals. Today, we know in mammals that P deficiency and hypophosphatemia (condition of low 

serum Pi) cause various disorders in terrestrial animals, including erythrocyte dys function, glucose intolerance, 

necrosis of skeletal muscle, myocardial dysfunction, central nervous system dysfunction, and osteomalacia 

(reviewed in Lotz et al. 1968; Knochel 1977; Kreisberg 1977; Berner & Shike 1988; Hodgson & Hurley 1993; 


10

Knochel 2000; Haglin 2001). These clinical symptoms are associated with speci fic metabolic alterations as 

follows. The decline of erythrocyte 2,3-DPG in P deficiency shifts the oxyhemoglobin dissociation curve to the 

left, causing hypoxia at the cellular level (Lichtman et al. 1971; Travis et al. 1971). The decrease of blood ATP 

level in P deficiency has been report ed in rats (Rapoport & Guest 1938), chickens (Bishop & Williams 1958) and 

humans (Lichtman et al. 1969, 1971; Travis et al. 1971). In mammals, chronic P deficiency decreases ATP 

concentration in skeletal and cardiac muscles as well as in kidney (Knochel, 2000). Day & McCollum (1939) and 

Hardy et al. (1993) reported lethargic condition of rats and fish, respectively, under P-deficiency, which might be 

related to the low ATP levels in blood or other tissues (these workers did not measure ATP levels). Mellanby 

(1919) also reported a similar observation, "Again, the rachitic puppy is lethargic and does not jump about; . . . 

Similarly, just as the rachitic baby is a good baby and does not cry much, so also the dog in this condition seldom 

barks or makes the superfluous efforts practiced by the normal healthy puppy." The decrease of serum or cellular 

Pi reduces mitochondrial respiration, glycogen, G6P and phospholipid-P in myocardium, skeletal muscle and/or liver. 

The low cytosolic Pi concentration impairs fatty acid oxidation, causing marked hypercholesterolemia (Horl et al. 

1983; Brautbar & Massry 1984; Brautbar et al. 1984; Kreusser et al. 1984). In humans, a low P intake 

increas es de novo synthesis of fats from glucose (Rudman et al. 1975). The anorectic effect of P deficiency may 

be related to an increase of reducing equivalent (Langhans et al. 1985) as a result of impaired oxidative 

phosphorylation. Hypophosphatemia lowers intracellular Pi concentrations, which impairs phosphorylation of G3P 

and thus the accumulation of G3P and decline of 1,3DPG and 2,3DPG, which impairs glucose utilization and ATP 

synthesis in glycolysis (Rose et al. 1964; Travis et al. 1971; Davis et al. 1979; DeFronzo & Lang 1980), increases 

insulin secretion in response to glucose (Marshall et al. 1978), and may cause hyperinsulinemia accompanied by 

carbohydrate intolerance (Harter et al. 1976). Chronic hypophosphatemia (P deficiency) thus elevates fasting 

blood glucose and insulin levels, increases lipogenesis, and reduces growth hormone (diabetogenic) secretion and 

protein synthesis. These metabolic shifts resemble to the etiology of obesity as studied in McCann et al. (1997). 

In fish, effects of dietary P restriction on metabolism have not been well-studied except that the increase of fats in 

the body of fish fed P-defi cient diets is a common observation (see Table 2). This observation agrees with Liebig's 

account mentioned above. Sakamoto & Yone (1980) analyzed fat and fatty acid in the body of red seabream fed 

diets with or without P. Based on the fatty acid profile of dietary fat and that of the fish body, the authors 

concluded that the fat retained by the fish during P-deficiency was dietary origin rather than from de novo synthesis. 

These authors also showed that lipid absorption was almost complete (98-99%) and was not related to dietary P 

levels. Takeuchi & Nakazoe (1981) reported that carp fed low-P diets had higher concentrations of nonpolar 

lipids, especially oleic and palmitic acids, in muscle, hepatopancreas and viscera that corresponded to the degree of 

P-deficiency in the diets. When these fish were starved for 3 wks, fish previously fed low-P diet did not decrease 

body fat, whereas fish received high-P diets reduced body fat to one or two-thirds. However, fish that had received 

low-P diets lost considerably more weight during 3 wks of starvation than fish received higher levels of P. The 

authors postulated that the beta-oxidation of fatty acids was inhibited by P-starvation. Onishi et al. (1981) studied 

the effects of dietary P levels on the activities of hepatopancreatic enzymes in carp. P-defi ciency markedly reduced 

the activity of pyruvate kinase, but tended to increase the activities of citrate cleavage enzyme, glutamate 

dehydrogenase and gluconeogenic enzymes (fructose 1,6 diphosphatase and phosphoenolpyruvate carboxykinase). 

The activity ratios of fructose 1,6 diphosphatase / phosphofructokinas e and phosphoenolpyruvate carboxykinase / 

pyruvate kinase increased. The authors suggested that, in P-deficiency, gluconeogenic activity is dominant and that 

fatty acid synthesis from amino acids may be accelerated. Also, the authors suggested that P-defi ciency activates 

recycling of pyruvat e to phosphoenolpyruvate (futile cycle), which decreas es the activity of TCA cycle and energy 

production by the glycolytic pathway. Thillart et al. (1980) studied effects of direct hypoxia (not due to 

P-deficiency). The authors found that placing goldfish in water of low oxygen concent ration for 12 h markedly 

decreased PCr in muscle and liver, and the energy charge, ATP/(ADP+AMP) in liver of the fish. Fish previously 

acclimated to hypoxia, however, had higher levels of energy charge in liver and red muscle. Hypoxia due to low 

oxygen in water is generally known to decrease ATP and other organic phosphate levels in erythrocytes in various 

freshwater fishes (Wood & Johansen 1972, Smit & Hattingh 1981). Dobson et al. (1987) reported an increase of 

Pi and a decrease of PCr in trout muscle after 10 seconds of exercise. These authors also reported large di fferences 

between pre-exercise and 10 min. of exercise in the concentrations of PCr, ATP, Pi, lactate, glycogen, and several 

glycolytic intermediates in trout white muscle. These studies indicate that many factors other than diets or 

nutrition could have profound effects on the concentrations of P compounds in the body. Since P depletion reduces 

the utilization of glucose, production of ATP and PCr, and resistance to anoxia, P loading (above normal) is expected 

to elevate these functions above normal. However, studies with human athletes have shown inconsistent results 

(reviewed in Kreider 1992). Tarr (1949) determined various P compounds and glycogen in the skeletal muscle of 

various fishes and the white rat. The author noted that the contents of phosphorylated intermediates, especially PCr, 


11

were extremely variable and normally lower than the values of rats, and this might be due to fish struggles and rapid 

breakdown of the compounds when sampling the tissue. The author scooped the fish from the tank without using 

anesthetic, and the fish were apparently captured from natural waters and kept in aquaria before use. McCartney 

(1968) attempted to prevent by dietary P loading the accumulation of glycogen in liver of brown trout fed a diet high 

in carbohydrate, and to facilitate the utilization of dietary carbohydrate. Several pathways in glucose metabolism 

require Pi. One of which is the rate-limiting step in glycogenolysis catalyzed by the enzyme phosphorylase, which 

is specific for the phosphorylytic breaking of glycogen to yield glucose-1-phosphate. Increased availability of Pi 

may also promote the catabolism of glucose before glycogen form ation. A casein-based semi-puri fied diet 

containing 20% maltose and 0.34%P (from casein) was supplemented with either 0.37%, 0.74%, or 1.48%P from 

NaH 2PO 4. The dietary P level, however, had no significant effect on the liver glycogen content or liver total P 

content of the fish after 22 weeks of feeding. McCartney (1969) repeated the previous experiment, but did not see 

a definite effect of dietary P loading on the liver glycogen level. Liver phosphorylase activity was slightly higher 

in fish fed diets of higher P content. Liver Pi level decreased with increas ed dietary carbohydrate level but dietary 

P level had no effect. Shimizu et al. (1969) noted that NADH dominates over NAD in muscle and liver of many 

fish species (caught from natural waters), and that this is a distinct difference from mammals. Takamatsu et al. 

(1975) found that the ATPase (EC 3.6.1.3) activity in myosin of the skeletal muscle of juvenile common carp was 

higher in fish fed low-P diet than those fed high-P diet. The authors did not discuss about this difference. The 

fish fed the low-P diet had higher body fat content than those fed the high-P diet. Since P-deficient fish reduce 

efficiency of energy or feed utilization without reducing the digestibility, the fish must have some altered 

metabolisms to "waste" dietary energy. Various tissues are known to contain phosphatases that hydrolyze 

high-energy phosphate bonds, causing net loss of the energy. Various futile pathways might also be activated in P 

deficiency. Such wasteful reactions would be more important shortly after a meal (heat increment ) or at other 

times when substrate concentrations are high (Hegsted 1974). The high ATPase activity of the P-deficient fish 

might therefore be due to diversion of the energy flow toward such wasteful pathways. Other energy-wasting 

pathways may also be responsible, including resynthesis of glucose from pyruvate through oxaloacetate (futile 

cycle), hydrolysis of triglyceride and its resynthesis, protein synthesis and breakdown. Another consideration is 

uncoupling of oxidative phosphorylation. The uncoupling protein shifts the energy released from the proton 

(NADH) toward thermogenesis and away from the ATP synthesis, meaning that the process does not require Pi. 

However, no proven evidence seems to exist in fish and higher vertebrates for such metabolic alterations under 

P-deficiency. Kleiber (1945-46) indicated, however, that when a heat loss is decreased by supplementing a diet 

with a nutrient, the diet is deficient in that nutrient. Thus, even though there is no data, P-deficient fish might be 

generating heat, instead of ATP, in the respiratory chain. In rats, Huber & Breves (1999) reported that P-depletion 

decreased the growth and feed conversion, but increased N-retention and decreased renal glutamate dehydrogenase. 

The authors also reported that P-depletion in rats did not affect lipid metabolism but reduced gluconeogenesis. 

Sugiura et al. (2000) noted that ATP, PCr, G6P concentrations in blood and skeletal muscle of post-juvenile rainbow 

trout were relatively constant after 21 days of dietary P restriction (or excess), whereas plasma and urinary Pi 

concentrations of the same fish responded rapidly to the dietary P level. 

Molecular responses to dietary P restriction 

Traditional indicators are clinical signs or symptoms, such as growth depression and bone malformation. Urinary, 

plasma and bone P levels are non-clinical indicators, which are sensitive but not very reliable due to large 

variabilities. Also, these and other non-clinical indicators bear different interpret ations (requirement vs. saturation 

as discussed above). The search for more sensitive and reliable indicators that can detect or even predict 

P-deficiency or suffici ency is important. P-containing intermediary metabolites studied so far, however, have been 

proven insensitive as the indicator of P status (discussed above). Molecular indicators could be sensitive 

prognostic tools to predict P-deficiency well before any clinical symptoms may arise. 

The type 2 NaPi transporters (NaPi-II) are the best-known P-responsive genes. The NaPi-II mediates 

transcellular Pi transport from the intestinal and renal tubular lumen across the apical membrane of the epitherial 

cells. In mammals, NaPi-II mRNA is upregulated in dietary P restriction; but it takes a few days (kidney) or a 

week (intestine) to respond to dietary P restriction. However, in the epithelia of renal proximal tubules, the 

trafficking of NaPi-IIa and IIc proteins appears to occur quite rapidly (within a few hours), which is independent of 

the gene transcription. The retrieval of renal NaPi-IIa protein from the apical membrane occurred only 2 h after 

consuming a high-P diet and 35 min after PTH injection via PKA and PKC signaling (Murer et al. 2000), and only 

15 min after increasing cerebrospinal fluid Pi concentration without a change in plasma Pi concentration (Mulroney 

et al. 2004). These rapid responses may not be specifi c to body P status, but rather a response to immediate dietary 


12

P intakes. Also, examining the trafiking of NaPi protein requires immunohistochmical analysis of the tissue 

sections, which is too laborious to perform routinely to diagnose body P status or adequacy of dietary P intake. 

Werner & Kinne (2001) reported phylogenetic relationship of NaPi-II speci es from fish to higher vertebrates. 

Interestingly, in fish both renal and intestinal NaPi are type IIb, whereas in mammals intestinal NaPi are type IIb and 

the renal isoforms are typeIIa. In fish, Coloso et al. (2001, 2003) showed that intestinal and renal NaPi-IIb genes 

are upregul ated by chlonic dietary P retriction. In trout pyloric caeca, NaPi-IIb mRNA abundance appears to be 

more responsive to dietary P compared with intestinal and renal responses, making it a potential indicator for P 

adequacy or deficiency. The quantification is relatively rapid and accurate by use of realtime RT-PCR. NaPi-II 

genes contain vitamin D response element (VDRE) as well as cAMP and estrogen-response elements in the 

promoter region (Xu et al. 2003). Therefore, the transcriptional activation of NaPi gene must be mediated by those 

factors. CYP27B1 (25-hydroxyvitamin D-1alpha-hydroxylas e) and CYP24 (25-hydroxyvitamin 

D-24-hydroxylas e) are two key cytochrome hydroxylase enzymes (P450) in vitamin D metabolism, and are partly 

regulated by dietary P to maintain P and Ca homeostasis in body. Low-P diets down-regulate CYP24 mRNA 

expression, thereby preventing the catabolism of the active form of vitamin D3, 1,25(OH) 2D3 (calcitriol) at the time 

of P defi ciency. 

The renal 1-alpha hydroxylase (CYP27B1) mediates hydroxylation of 25(OH)D to form 1,25(OH)2D, the 

most potent metabolite of vitamin D. P deprivation is known to stimulate CYP27B1 activity (Tanaka & DeLuca 

1973, Portale et al. 1984, Condamine et al. 1994). In the rat, Yoshida et al. (2001) reported that feeding low-P 

diet caused 4 fold increase of CYP27B1 protein and 10 fold increas e of CYP27B1 mRNA in the kidney after 4 days 

of dietyary P restriction. In young rats, Lai et al. (2003) reported 3-fold increase of CYP27B1 mRNA at d3, but 

not d5 of dietary P restriction. In adult rats, the mRNA expression level was very low and there was no significant 

increas e by dietary P restriction. Zhang et al. (2002), however, showed sustained increase of CYP27B1 mRNA 

from d2 to d8 on a low-P diet. In young rats, Katai et al. (1999) and Xu et al. (2002) reported that intestinal NaPiII 

mRNA abundance and Na+-dependent P transport rate increased aft er cal citriol injection. Dietary P deprivation 

increas ed CYP27B1 mRNA abundance in rat kidney (Yoshida et al. 2001) and stimulated CYP27B1 activity to 

increas e calcitriol synthesis (Portale et al. 1984; Condamine et al. 1994). These results in mammals show the 

regulatory role of cal citriol on NaPiII transcription and suggest that the expression of CYP27B1 is critical to NaPiII 

regulation. In fish, CYP27B1 sequences rem ain to be identified. Several fish EST sequences that are moderately 

homologus to mammalian CYP27B1 are identified; however, they are equally homologus to CYP27A1. CYP27A1 

also has some 1-alpha hydroxylase function (Araya et al. 2003). In birds, Nemere (1996) reported that vascular, 

but not luminal, perfusion of calcitriol rapidly increas ed intestinal Pi uptake. In fish, however, incubating renal 

proximal tubule cell cultures of flounder (Lu et al. 1994) or intestinal sleeves of trout (Avila et al. 1999) in a 

solution containing calcitriol did not alter net Pi transport rate. In trout, Coloso et al. (2003) reported that plasma 

calcitriol concentration did not change after chronic moderate dietary P restriction. These results in fish suggest 

that the role of vitamin D in P homeostasis may be different between fish and mammals. In mammals, other 

mechanisms by which dietary P regulates NaPiII expression also include hypophosphatemic proteins that alter 

NaPi-II mRNA stability (Markovich et al. 1995; Moz et al. 1999), coregulators that alter the rate of NaPi-II 

translation (Moz et al. 1999), and a P-response element that increases the rate of NaPi-II transcription (Kido et al. 

1999). In fish, little is known regarding the molecular mechanisms of NaPi-II expression; however, since most 

dietary P in fish is absorbed via paracellular di ffusion, the contribution of NaPi-mediated dietary P absorption at 

normal dietary P intakes is insignificant compared with other factors, including the forms of phosphate compounds, 

chemical interactions and luminal environment in the intestine, all of which appear to have more decisive effects on 

biological availability of dietary phosphorus in fish. 

The 24 hydroxylase (CYP24) is the first enzyme in the catabolic pathway for vitamin D compounds 

(Omdahl et al. 2002). Wu et al. (1996) reported that rats fed a low P diet showed a five-fold decreas e in renal 

CYP24 mRNA compared with rats fed a normal P diet. CYP24 mRNA and CYP24 activity decreas ed within 24 h 

of Pi restriction, reaching a minimum by 48 h, and remained low through 14 days. Higher Pi (1.2% P) did not 

increas e CYP24 mRNA over normal P diet. Zhang et al. (2002) showed that CYP24 mRNA was down-regulated 

by dietary P-restriction in a dietary P-level-dependent manner, and the decrease of the mRNA level by low-P is 

sustained from 1d until the end of study (d8). In trout, mRNA expression of renal CYP24 appears to be acute but 

transient in dietary P restriction. 

The vitamin D receptor (VDR) is a zinc-finger transcriptional factor regulating the expression of several 

genes. The VDR, once liganded with 1,25(OH) 2D (calcitriol), forms a heterodimer complex with retinoid X 

receptor (RXR). The VDR/RXR complex recognizes a special DNA sequence called vitamin D response element 

(VDRE) that are pres ent in the promoter region of vitamin D-regulated genes, including NaPi-II. When cofactors 

and regulators are available in the cell, VDR/RXR complex can initiate transcription of the genes. In chickens and 


13

ats, Meyer et al. (1992) and Sriussadaporn et al. (1995) found that low dietary P up-regulates intestinal VDR 

mRNA abundance in the intestine. The increased gene expression, however, was modest in magnitude, brief in 

duration and limited only to intestinal tissues. Fish VDR mRNA sequences have been identified in zebrafish and 

flounder; however, the functional roles have not been studied. In flounder, VDR mRNA appears to be omnipresent, 

but it may be absent in liver (Suzuki et al. 2000). In chickens, however, a trace amount of VDR mRNA was 

present in liver (Lu et al. 1997). In trout, VDR mRNA appears to be distributed in various tissues. In trout, 

intestinal VDR mRNA abundance was apparently independent of diet at all times, while renal VDR mRNA 

increas ed significantly at d5 (~2 fold) but not at d2 or d20 of dietary P restriction. Segawa et al. (2004), however, 

reported that VDR-null mice increased intestinal NaPi mRNA and protein expressions as well as Pi transport in 

dietary P restriction, suggesting that intestinal NaPi-II expression is also mediated via a mechanism that is 

independent of the vitamin D-signaling. 

Phosphatonins are hypophosphatemic factors that cause phosphaturia in mammals, which include fibroblast 

growth factor 23 (FGF23), frizzled related protein 4 (FRP4), and matrix extracellular phosphoglycoprotein (MEPE). 

FGF23 is highly expressed in certain tumors (tumor-induced osteomalacia: TIO) and in FGF23 gene mutations 

(ADHR) (Takeda et al. 2000). FGF23 may be the (indirect) substrate for PHEX that is expressed in hard tissues. 

Thus, in patients of X-linked hypophosphatemia (XLH), who have a PHEX gene mutation, FGF23 accumulates in 

circulation, causing renal P wasting. In mammals, patients of chronic renal failure (accompanied by 

hyperphosphatemia) show elevated circulating FGF23. FGF23 suppresses CYP27B1 expression in kidney, and 

both NaPi-IIa and IIb expression, causing reduction of Pi absorption in both kidney and intestine. 1,25(OH) 2D3 

injection increases serum Pi and FGF23. Serum Pi and FGF23 concentrations appear to be highly and positively 

correlated one another. There is presently no sequence in fish EST databases that shows significant homology to 

mammalian FGF23. Thus, the roles of FGF23 in fish remain to be studied. 

Custer et al. (1997) found another gene that was up-regulated by ~2 fold in dietary P restriction, which they 

tentatively called diphor-1 (currently PDZK1). PDZK1 and other PDZ proteins such as NHERF1 appear to 

participate in the apical-cytosolic traffi cking of NaPi-IIa protein in mammals (Biber et al. 2004). PiUS is an 

inositol hyxakisphosphate (IP6) kinase, which transfers Pi from diphosphoinositol pentakisphosphate (PP-IP5) to 

ADP to form ATP. Norbis et al. (1997) identified a cRNA in rabbit small intestine that stimulates Pi uptake when 

injected into Xenopus oocytes, which they tentatively called inorganic phosphate (Pi)-uptake stimulator or PiUS. 

Subsequently, PiUS mRNA was found to be upregulated about two folds in rat intestine after 7 days of dietary P 

restriction (Katai et al. 1999). In trout, however, PiUS mRNA in the intestine and kidney appears to be insensitive 

to dietary P restriction. 

Mitochondrial Pi carrier protein appears to be up-regulated (~3-fold) in dietary P-restriction in rainbow trout. 

This protein transfers Pi from cytosol to mitochondrial matrix for oxidative phosphorylation. Thus, up-regulating 

(increasing) this protein might be necessary to supply Pi to the respiratory chain under limited cytosolic Pi 

concentrations. In mammals, acute hypophophatemia, typically associated with glucose perfusion, causes the so 

called Crabtree effect that reduces ATP production due to competition between glycolysis and oxidative 

phosphorylation under limited cellular Pi availability (Brazy & Mandel 1986, Brazy & Chobanian 1996). 

Chronic hypophosphatemia is also known to be associated with decreas ed erythrocyte ATP concentration in animal 

species (Knochel 1977). In trout, however, blood ATP concentration seems to decreas e only slightly and muscle 

ATP changes little by chronic (24 d) dietary P restriction, which might be due to the up-regulation of mitochondrial 

Pi carrier protein. 

Kuro-o et al. (1997) found a single gene that is associated with several aging-related syndromes. They 

named the gene klotho gene. Klotho (Clotho) is a Greek goddess who spins the thread of human life. The length 

of the string will determine how long a certain person's life will be. Klotho gene is expressed predominantly in 

kidney and brain. Interestingly, Yoshida et al. (2002) found, in klotho -/- mice, that serum 1,25(OH) 2D was 

markedly elevat ed; renal CYP27B1 mRNA was also markedly upregulated; renal CYP24 mRNA was slightly 

upregulated, serum P and Ca were also elevated; serum CT was slightly elevated; and serum PTH was slightly 

decreased compared with wild-type mice. Morishita et al. (2001) found in klotho mutant mice that low-P diet 

prevented the progression of senes cence, and even induced the expression of klotho protein. Also, in wild-type 

mice, high-P diet decreased renal klotho protein expression. 

Fish have no parathyroid gland; however, Gensure et al. (2004) has identified, in zebrafish and pufferfish, 

PTH/PTHrp (parathyroid hormone-related peptide) receptors. These investigators also identified two different 

PTH in zebrafish that bind to these receptors. The PTH genes are reported to be expressed in the lareral line 

(embryo, transient) and in the brain and neural tissues (only PTH1). The physiological roles of fish PTH, however, 

remain to be studied. Calcitonin (CT) is a hypocalcimic hormone in mammals, which also appears to affect P 

homeostasis. CT decreases blood Ca level by decreasing Ca absorption in the intestines, by decreasing Ca 


14

eabsorption in the kidney, and by decreasing osteoclast activity. In fish CT is secreted from the ultimobrachial 

glands, but the role is not well-defined. Wagner et al. (1997) reported that salmon CT inhibited gill Ca uptake in 

trout. Stanniocalcin (STC) prevents hypercalcemia in fish by inhibiting gill Ca uptake. STC also stimulates renal 

Pi reabsorption in fish (Lu et al. 1994). Lu et al (1994, 1995) reported a wide variety of factors regulating P 

metabolism in fish using flounder renal tubule cell cultures, where net Pi reabsorption was increased by salmon 

stanniocalcin, rat prolactin, salmon/flounder somatolactin, bovine PTH (bovine), and salmon growth hormone. In 

mammals, STC decreases net Ca transport and increas es net Pi transport in the intestine, increases Pi reabsorption in 

kidney, and increases Pi uptake in osteogenic cells (via type-3 NaPi). Mammalian STC appears to have many other 

functions (Ishibashi & Imai 2002, Yoshiko & Aubin 2004). In rats, Cheung et al. (2002) reported six P 

responsive genes, including beta-actin, in the renal proximal tubules based on proteomics. 

Indicators of P status: Perspectives 

Some molecular indicators appear to be more sensitive and reliable than traditional indicators to diagnose body P 

status and to predict potential dietary P deficiency. However, as discussed above, molecular indicators are 

relatively difficult to analyze, making their practical use limited. But, do we need to analyze anything? In the 

near future, P status of fish may be “ expressed” by the fish by way of their body color, for eample. A recent study 

on plant P-responsive genes suggests that P responsive genes may be modified (Hammond et al. 2003, 2004). 

Such genes may contain a P-response element, such as vitamin D response element, in the promoter region upstream 

of the transcription initiation site. Then, the P-responsive promoter is fused to a reporter gene, such as melanin 

(instead of luci ferase), and inject it back into the original fish (embryo). The expression of such genes may be 

tissue or cell specific. Where the cell type is appropriate, the fused marker gene will be turned on when dietary P 

intake is inadequate, increasing the transcription of melanin mRNA, making the fish darker in color. Such marker 

fish may eventually replace all laboratory analysis. It will be necessary, however, to identify the P responsive gene 

in the skin, unless P-responsive gene from other tissues can be adequat ely expressed in the skin. Also, the mRNA 

to protein translation may be regulated. When fish look dark in color, simply increase dietary P a little, and when 

they look normal, try reducing dietary P until some of them become dark. A fine adjustment of dietary P 

concentration can be done by using two diets, one slightly P-defi cient and another slightly P-excess, and mixing 

them at an optimum ratio based on the fish color. This will be particularly useful since, as mentioned above, 

dietary P requirement varies greatly depending on many factors, including feed quality, developmental or 

physiological stage, and cultural conditions. 

P requirement for large or adult fish 

First of all, we should consider the reasons why there is no data on large fish. The most important reason is that 

large fish are quite resistant to nutrient deficiency compared with small fish, and thus they do not respond to dietary 

restriction of nutrients if conventional response indicators are used. My calculation (based on body P reserve and 

shifting feed effi ciency during the life stages) indicates that when small fish (1g body weight) are fed P-deficient 

diet containing 0.2% available P, their growth will be arrested in seven days on the diet. For fish of 10 g body 

weight, it takes 20 days before their growth is depressed. For fish of 500 g body weight, it takes 232 days of 

feeding, and for fish of 1 kg, their growth will never respond to the diet. Major factors responsible to these 

differences are that: (1) large fish may have large initial body P store, (2) large fish have lower feed efficiency than 

small fish, and (3) large fish have lower feeding rate per body weight. Therefore, it is probably impossible to 

accurately determine dietary P requirement for large fish based on growth rate. This does not mean that large fish 

can be reared on P-deficient feeds because their initial P store is not known. Thus, it is important to know the P 

requirem ent for large fish to minimize the risk of P-depletion (see section “Phase-feeding and finishing feed”). 

Satoh et al. (1987) wrote, "Fish weighing over 2 g are not suitable as experimental animals for determination 

of mineral requirements in terms of diffi culty to induce mineral defici ency." This is probably true; however, it is 

still necessary to know the nutrient requirements of large fish regardless of the diffi culty, as Cowey (1995) states, 

"Since about 90% of the food used in fish farming is given to large fish (100 g

environmental standpoint, studying the precise dietary P requirement for large (1kg) fish is 32 times more important 

than studying the same for small (10g) fish. When fish are grown larger than 1kg in size, the importance of 

studying the requirement for those fish will become much higher. When the initial fish size is large, however, it 

will be difficult to magnify the size relative to the initial. In order to achieve a satisfactory level of growth 

magnifi cation, large fish must be fed for a much longer period since their speci fic growth rat e is much lower than 

their young as in other animal species. Additional problem of keeping large fish for such a long period is that the 

amount of feed they consume should be much more than the amount required for young fish. This makes the use 

of a semi-purifi ed diet economically unaffordable in many cases. Ironically, one of the most important discoveries 

that have been made in the history of nutrition or physiology research is the use of small or young animals such as 

rats and mice (for example, Lavoisier used the guinea pig in a respiration study). Small or young animals grow 

rapidly but consume only a small quantity of food, which thereby made the use of purifi ed (nutritionally defined) 

diets possible. Nutrition research for large animals, including fish, therefore, may require unconventional 

approaches and techniques that differ from those used for small or young animals. 

Feeding duration and the fish size will have a profound effect in estimating dietary P requirements, especially 

when the initial fish size is large. However, errors from such sources cannot be eliminated completely. Thus, it 

will be important to verify the accuracy of estimated values. One way to do this is to determine the requirement at 

intervals (e.g., alternate weeks) until the values stabilize. Body pool size or diet history is an important source of 

error when working with large fish and with relatively short feeding duration. Gillis et al. (1953) fed laying hens 

with diets of varied P levels for 35 weeks. P-deficiency did not reduce egg production until after 12 weeks, 

suggesting that at least 12 wks of feeding is necessary when estimating P-requirement based on egg-production. 

Singsen et al. (1962) followed 40 weeks to monitor responses of laying hens to varied dietary P intakes. The 

dietary P level did not affect the egg production until after 8 weeks. Laying hens and lactating cows have been 

studied for their P requirement based on egg or milk production; however, in fish culture, neither of them is more 

important than fish growth. Skonberg et al. (1997) tested only 5 dietary P levels, and did not extend the feeding 

beyond 8 weeks. However, their data at wk-4 show that P and Ca contents in fish body and skin increased linearly 

with the dietary P intake (i.e., no response plateau), whereas at wk-8 fish on high-P diets reached apparent plateaus 

in those measurements. It is uncertain, however, if the plateaus (break points) at wk-8 could remain constant or 

decrease further i f the fish had been fed for a longer period. Rodehutscord (1996) determined Ca and P contents 

of fish per gain rather than per fish body by correcting the background (Ca and P contents of fish at the beginning of 

experiment). This procedure, however, does not eliminate or reduce problems described above since the size of the 

initial body pool is still variable from fish to fish, which buffers gain or loss of nutrients during growth. Body 

pool size or diet history is an important source of error when working with large fish and/or with relatively short 

feeding duration. To reduce this source of error, determining the requirement more than once in the course of a 

feeding period will be required. Aternatively, it will be more convenient to use highly responsive indicators to 

examine the adequacy of dietary P intake in large fish. Several workers have shown in mammals and fish that 

urinary (or non-fecal) P output is a sensitive indicator for P. The urinary response is very fast (1-3 days), clear, 

consistent and less vulnerable to diet history, demonstrating the distinct advantage of monitoring urinary P to 

determine dietary P requirement for large fish. In addition, since urinary P is the most problematic source of P in 

effluent, establishing dietary P level that can minimize urinary P is itself rational from an environmental standpoint. 

Using the urinary method, however, is not applicable for other important cases such as diagnosing fish on site 

(aquaculture ponds). Thus, it is important to explore alternative methods, such as molecular indicators (described 

before). 

Balance technique 

The balance method, as McCay once stated, is "similar to the trade balance between nations". It is an old method 

to estimate dietary nutrient requirements, and is still used today with little or no revision. Boussingault (discussed 

below) pioneered in the balance trial, especially of nitrogen and fats, using livestock animals. The results were 

reported in his famous book entitled "Rural Economy". He used the balance method to study the possibility of N 

fixation (from air) by animals, and also fat de novo synthesis (from CHO) by livestock animals. These are both 

landmark nutrition studies in history, but not discussed here any further. Almost 50 years before Boussingault, 

however, Vanquelin (1799) conducted a balance experiment using hens. He measured Ca phosphate, Ca carbonate 

and silica in the food they consumed, in the eggs they laid, and in the excrement during 10 days. His calculation 

showed that the excretion was much larger than the intake in the quantity of Ca phosphate. He concluded that a 

transformation of other elements into Ca had occurred in the metabolism of hen. He did not recognize that bones 

served as storage centers. This conclusion, however, was accepted by his contemporaries. Boussingault (1845) 


16

eported the results of bal ance experiments on mineral constituents of food. He measured Ca, P, Mg, sulfur, 

chlorine, sodium, potassium, iron, aluminum, silica, ash, dry matter and nitrogen in the hay, excrements and urine of 

a cal f, and estimated the amounts of these substances retained by the animal. The amounts retained by the animal 

were considered the references to the minimum requirements, which must enter into the constitution of the food. 

Boussingault (1846) also studied the development of the skeletal system of the pig by analyzing the contents of 

Ca-phosphate, Ca-carbonate, Mg, phosphoric and carbonic acids, dried skeleton and ash in the body of the newborn, 

juvenile and adult. Based on these data, he estimated the minimum amount of Ca-phosphate which the diet of a pig 

must provide for normal skeletal development. Day's book published in 1860 contained a chapter entitled 

"Nutrition" (Chapter xx), and a section "Digestibility” (in Chapter xix). In the Nutrition chapter, he wrote, " . . . 

these four essential adjuncts (the protein-bodies, the fats, the carbo-hydrates, and the mineral matters) in the 

metamorphosis of the animal body must also be contained in those articles of food which are required for the 

renovation and restration of those particles which have been lost or worn out, and for the due accomplishment of the 

vital phenomena (p. 487)." Here the basic concept of the balance method is apparent. Weiske (1873) fed Ca 

phosphate to calves, and estimated the daily requirements of Ca and P based on the amount of these elements 

retained. Forster (1873) made a dog in near equilibrium in P by feeding a low-ash ration, and estimated the 

minimum requirement. Cronheim analyzed P, Ca and K contents in the flesh of carp at the age of yr-1, 2, and 3. 

The data showed that the contents of Ca and K were similar among the age groups, but that of P tended to increase 

as the age of the fish increas ed. He suggested that the diet should contain 3 times as much of these minerals as the 

fish retain (cited in Higure 1912). Kellner (1909) estimated the dietary requirements of Ca and P for calves and 

pigs from the balance dat a and by allowing 2-3 times as much Ca and P in the food as the animal will store in the 

body. The balance method is still common in human nutrition in estimating dietary requirements of nutrients 

including P (IOM 1997). IOM (1997) also applied factori al approach to estimate dietary P requirement for 

humans under the ages of 18 years. The factors they included in the calculation were P accretions in bones and soft 

tissues during growth, urinary loss, and the efficiency of absorption. However, they also mentioned that a better 

way in estimating the requirement for this growing age group is the use of a balance plateau, which is the estimation 

for the intake that maximizes body P retention. The body P retention is measured by either balance (in humans) or 

direct body analysis (in animals). Although the balance method is so commonly used, Hegsted (1976) and Mertz 

(1987) are critical for its use in estimating nutrient requirements. They argue that the balance method simply 

measures the amount of a nutrient that is needed to maintain the size of the existing body pool for that nutrient. It 

may be necessary, in balance methods, to study animals of different diet history with varied body pool sizes. 

Body P content 

Anderson (1878) presented numerous data of P content (and major bases) in various tissues (tendon, skin, kidney, 

lung, brain, heart, aorta, and spleen) in various animal species (ox, pig, sheep, human) under normal and diseased 

states. The author compared the differences in P content and P/base balance among different organs and species. 

He also presented data of P content in healthy urine and feces of humans consuming different diets. The author, 

however, did not directly study the effect of different P intakes on tissue P contents. Gibson & Estes (1909) 

analyzed salmon tissues for total P content by both colorimetric and gravimetric methods following acid digestion. 

The data showed that P content of skin was higher than that of liver, muscle, testes or gut on an entire tissue basis. 

Skin contained P in the amount ca. 5 times more than muscle. The authors did not analyze the bones. Takeuchi 

(1915) analyzed fish muscle for fat -soluble P, water-soluble P, insoluble P, inorganic P (Pi), and total P. In both wet 

and dry basis, inorganic P content in muscle of carp was lower than that of flounder, 2 species of tuna, bonito, 

bluefish (mutsu), and shark. Total P in muscle of these species was about 0.5% (wet basis) or 2.0% (dry basis) of 

which 60-75% were inorganic P and the remaining fractions were mostly phospholipids. Phillips et al. (1953) 

reported Ca, P, and Mg contents of the whole body and various tissues in the body of brook trout at different sizes. 

The head contained the highest percentages of both Ca and P than the other parts. Phillips et al. (1964) reported 

that skin is one of the important repository sites for the absorbed 32 P from diets. Distribution of total P in the body 

of trout was 39% in the bones (skeleton), 28% in the skin, 2% in the GI tract, and 31% in the muscle, and the total P 

concentration in this order was 17.0, 8.8, 2.3, and 1.9 mg per g of tissue. Podoliak & Smigielski (1971) had 

different values: 18% in the bones (skeleton), 22% in the skin, 3% in the GI tract, and 57% in the remainder sample, 

and the total P concentration in this order was 14.2, 7.3, 4.1, and 3.6 mg per g of tissue. Two days after feeding a 

diet containing 32 P, skin contained about 1/4, the bones 1/6, and nearly 1/2 of the recovered label ed P was in the 

remainder samples of the body. Cowey (1995), however, concluded that the main P reservoir in fish body was the 

fl esh with skeleton (head, skin, bones and tail) and other tissues collectively containing only about a quarter of that 

present in the flesh. The conclusion appears to be drawn based solely on the data of Chang et al. (1960) with 


17

maturing salmon. Chang et al. (1960) in his paper neither discussed about their data of low P levels in skeletal 

tissues nor compared their values with data reported by others (e.g., Shearer, 1984; Poston & Ketola, 1989). The 

data showed that the total P concentration (µg P/g tissue) of the head (with skin, bones and tail) was only about 1/10 

to 1/5 of other parts of the body (flesh, gonads, GI tract, liver, or kidney). In humans, skeleton is the storage site of 

P and Ca, and skin seems to serve as an irretrievable store of Pi (Anderson & Barrett 1994). Satoh et al. (1984) 

analyzed the content of ash, Ca, P and other minerals in the whole body of tilapia during 82 days of starvation (at 25 

and 15°C). During this period, fish weight decreased from 43.8 to 37.5 g, body protein from 16.5 to 13.6%, body 

fat from 8.3 to 2.9%, while body moisture increased from 70 to 77%. Ash, Ca and P contents (per wet whole body) 

all increased, since the fish became skinny or bony on starvation. The Ca/P ratio in the whole body increased 

during starvation, suggesting that fish lost muscular tissues that contained P but only trace amounts of Ca. 

The body fat content is quite variable depending on the nutritional status and also on the age of the animal, 

but total of fat and water contents in body is relatively constant since the amount of fat is inversely related to the 

amount of water in the body (Kleiber 1975; Papoutsoglou & Papoutsoglou 1978; Bondi 1987; Shearer 1994). 

Body mineral contents, therefore, should be expressed on a wet body basis (not dry body basis). Shearer (1984) 

showed that whole body P content of rainbow trout of various sizes (life stages) is much more constant when 

expressed as wet body basis than as dry body basis. A similar observation was reported by Satoh et al. (1987) with 

rainbow trout. They also reported that Ca and P contents in fish body were higher when diets were not 

supplemented with Zn than when they conteined supplemental Zn. In fish, body P concentration ranges between 

3.8 and 4.5 g per kg body weight with a few data below 3.0 and above 5.0 depending on the fish P status (reviewed 

in Wiesmann et al. 1988, Lall, 1991). Satoh et al. (1996) showed that whole body P of rainbow trout increased 

from 0.41 to 0.50%, which corresponded to the dietary P level (5 graded levels from 1.2% to 3.0% P in mostly 

available form; Note that the dietary requirement is only ~0.6%). In contrast, the vertebral P content decreased 

from 10.3 to 8.7% (fat-free dry basis) as the dietary P level increased. According to Bondi (1987), the proportion 

of each mineral, when expressed as percentage of “ fat-free dry body substance”, seems to be very similar among 

species in adult mammals. 

Factorial approach 

The factori al approach has been discussed by some workers to 'estimate' the dietary P requirement in fish. Pfeffer 

& Pieper (1979) derived the requirement value from: Gross requirement = (Amount retained + Endogenous 

loss)*100 / Availability. Shearer (1995) considered Dietary requirement = Gross requirement / Feed consumed. 

Nakashima & Leggett (1980) used the following formula to estimate P surplus (=urinary P excretion): P surplus = P ingestion 

(food) – P growth – P egestion (feces) – P maintenance. From this, the dietary P requirement (P ingestion when P surplus is 0) = 

P growth + P maintenance + P egestion. The amount retained by the fish and the availability can easily be found by analyzing 

body P content and by measuring the absorption. The most uncertain factor in the above formulas is the 

endogenous loss or the maintenance requirement. The first researchers measured the endogenous loss using 

starving fish with some reservation that the data of starving fish might be too low due to "sparing mechanisms" by 

fasting. The second researcher used the fi rst researchers' data. The third researchers used data of Kitchell et al. 

(1975) who determined the maintenance requirement of P for juvenile bluegills to be 5.4mg P/g body P per day. 

The calculated values by the third authors for juvenile yellow perch, however, showed that the requirement for 

maintenance was higher than the requirement for growth (ret ention). This is unlikely if fish are fed normally and 

growing normally. P-requirement for maintenance (endogenous obligatory loss) may not be determined on 

starving fish since starvation is a catabolic state. Growing fish require minerals, but starving fish do not. The 

obligatory loss of P is lower during growth than during fast, which is generally known in higher animals and humans 

(see Part 1, Endogenous loss). The obligatory nonfecal P loss by rainbow trout fed P-deficient diet was report ed 

undetectabl e (Sugiura et al. 2000). Theoretically, fish lose P in an amount that corresponds to obligatory N loss 

from the wasting muscle during starvation. Fecal obligatory loss is also negligible compared with dietary P intakes 

becaus e the apparent availability of highly available P sources such as sodium or potassium phosphates is 95-98%. 

If the obligatory fecal loss is significant, the apparent P availability cannot be high. The above figures indicate that 

only up to 2-5% of dietary P (at near requirement level) may be attributed to the obligatory endogenous loss. This 

level will be lower if the true absorption of the P compound is lower than 100%. If the apparent absorption is used 

in the above formulas to calculate the requirement, the fecal obligatory loss must be ignored and only the nonfecal 

loss will be the factor of argument, which is, as mentioned above, close to zero. Schwarz (1995) wrote that the 

factori al deduction of requirements for minerals can provide only approximate requirement values, and that the 

dose-respons e technique is the best option to estimate requirements of minerals. 


18

Dietary requirement of organic P compounds 

Meischer (1896) and Paton (1897-98) determined the amounts of various P compounds (lecithin, Pi, and the P in 

nuclein and pseudonuclein) in muscle, ovaries and testis of salmon caught at estuary and upper water. The results 

convinced the authors to conclude that the P stored in muscles as simple phosphates is transferred to the ovaries and 

testes and there built up into organic combinations. Unfortunat ely, the authors did not measure P in the bones. 

Milroy (1908) conducted similar studies on the herring. These workers tried to uncover then unidentified 

precursors of the organic P compounds in gonads that increase rapidly during reproductive period. McCollum 

(1909) in the rat and McCollum et al. (1912) in the laying hen have proven that all organic P compounds can be 

synthesized from inorganic P in the diet. Thereaft er, other workers showed in other animals that all organic P 

compounds required by the body can be synthesized de novo from inorganic P. However, McCollum et al. (1939) 

wrote, "There is as yet no experimental proof that inorganic orthophosphates could serve the requirement of all 

species irrespective of age and degree of development." With this context, the discovery of Kanazawa et al. (1981, 

1983) is of particular importance. They found that the larval fish of red seabream, ayu, flounder, and knifejaw 

required dietary phospholipids (especially phosphatidyl choline and inositol) for growth, survival, and normal bone 

development. Poston (1990, etc.) in Atlantic salmon and rainbow trout, and Radunz-Neto et al. (1994) and 

Geurden et al. (1995) in carp also demonstrated dietary essentiality of phospholipids or lecithin during the 

first-feeding period. The essential components of lecithin are choline, Pi, fatty acids and glycerol. It should be 

noted, however, that choline and Pi (especially, available P) are both highly soluble. If these essential nutrients are 

supplemented to a diet as such, the leaching loss must be quite large and rapid, especially from microparticulate 

diets used for larval fish rearing. What fish are consuming may be different from what the investigators are feeding. 

P in microparticulate diets may be lost very quickly. The effect of phospholipids therefore could be due to its lower 

water solubility than sodium or potassium phosphates, rather than its nutritional essentiality. This possibility may 

be difficult to ignore. If what fish are actually consuming is different from what the researchers are tossing into 

fish tanks, we might need to re-study the dietary requirements of highly soluble nutrients, including P. 

Availability vs. Unavailability 

Many investigators concluded that dietary Ca levels do not affect dietary P requirement of fish (e.g., Ogino & 

Takeda 1976, 1978, Watanabe et al. 1980, Lovell 1978). However, since dietary Ca reduces intestinal P 

absorption (availability) as will be discussed later (Part 2), it should increase the dietary P requirement. The 

discrepancy might be due to insensitivity of the measurement (e.g. weight gain, retention) in the requirement studies 

compared with intestinal absorption in digestibility measurements. For example, when the net intestinal absorption 

of P is 99% in one diet and 95% in another diet, the difference between these two diets may be insignificant and 

non-detect able when growth, bone analysis, retention or digestibility per se is used as the response of fish since 

other factors such as feed intake, rearing conditions, and within treatment (experimental) errors affect these 

measurem ents as well. However, in the same experiment, if fecal P content of the fish is compared between these 

two diets, it will be 1% (per P consumed) in the first diet and 5% in the second diet. This means that the difference 

is 5 fold, and one should conclude that these are very different. Using the same data set from the same experiment, 

it is possible to say "little difference" in availability when there is a marked difference in unavailability, and vise 

versa. 

Absorption of waterborne P 

Sekine (1929) reported results of an experiment conducted in 1921. He found that the contents of inorganic 

constituents in chum salmon increased steadily from egg (eyed stage) to larva (with yolk) and to juvenile (before 

starting to feed). He wrote that this change was a clear evidence of the absorption of inorganic salts from the 

ambient water. Alcohol-soluble P also increased from the egg stage to the larval stage. He thought that this was 

due to the formation of lecithin-like phospholipins. McCay et al. (1931, 1936) found that trout fed diets low in Ca 

and high in P grew normally for many months, and that supplementing the diet with Ca did not alter the Ca and P 

contents in the trout body. Mullins (1950) noted that stickleback absorbed 32 P in smaller amount than 42 K from 

water through the gill membrane. Phillips et al. (1954) observed that starving brook trout absorbed both 45 Ca and 

32 P from water, but the absorption of the former was much higher. Yoshii et al. (1955) reported that 32 P was taken 

up from water by goldfish but it reached a plateau in ca.10 hours after placing the fish in the water and remained 

constant until 268 hours at which time the experiment was terminated. The 32 P level in fish reached close to the 32 P 


19

level in the water but did not exceed it. The fish absorbed 45 Ca readily and concentrated it 100 times the level in 

the water. When the fish were fed algae containing these radioactive minerals, the fish absorbed 32 P more than 45 Ca 

from the algae. Phillips et al. (1958) observed that brook trout absorbed about 400-1000 times more Ca than P 

from water. Increasing P concentration in water proportionately increas ed the quantity of P absorbed, while 

increasing Ca concentration in water did not increase the amount of Ca absorbed by the fish. Phillips et al. (1954, 

1958, 1959) noted that the labeled P that had been absorbed from the water was included in tissues of the GI tract 

(highest in pyloric caeca) and gills of the fish. Phillips et al. (1959) reported that labeled P absorbed from food 

was distributed mainly to muscular and bony tissues of the fish. Increasing dissolved P in water decreased the 

utilization of P from the food, whereas dissolved Ca increased the utilization of dietary P. Phillips et al. (1961) 

showed that the absorption of waterborne P by brook trout was only trace in quantity compared with the absorption 

from food. Ogino & Yasuda (1962) reported that larval rainbow trout markedly increased body Na, K and Ca 

contents after hatching (but before the first feeding), while P, Mg, Fe and Cu remained relatively unchanged during 

the same period. Lall & Bishop (1979) reported that rainbow trout reared in freshwater for 12 weeks had higher 

Ca and P and lower Mg, Na and K contents in the body than those reared in seawater with the same diet for the same 

period. Shearer et al. (1994) reported similar observations in Atlantic salmon. Mol et al. (1999) also reported 

that catfish reared in water low in Ca and/or Mg had higher body P and Ca content than those reared in water high in 

Ca/Mg. This suggests possibility that water-borne Ca/ Mg may depress intestinal P absorption of the fish. 

Expressing P requirement 

Estimated values of dietary P requirement, however, can vary greatly depending on (1) feed efficiency of the diet 

(thus, energy density, digestibility of major nutrients, nutrient balance, etc.), (2) availability of P in the diet (thus, 

dietary Ca level and acidity), (3) growth velocity of fish (thus, fish size and physiological state), (4) feeding duration 

(growth magni fication), (5) diet history (initial body P reserve), (6) response criteria, and (7) statistical methods used. 

Reporting or interpreting dietary requirement values out of this context is misleading. Probably there is no such 

value like “ dietary requirement”. The requirement is only for growth, reproduction, disease resistance, and other 

physiological needs. The diet is merely a vehicle of the nutrient. The amount of P required in a diet changes 

every time the composition of the basal diet changes. In this review, therefore, the values of “dietary P 

requirem ent” were not listed. (see Shearer 1995, Davis & Gatlin 1996 for tabulated requi rement values for P and 

other minerals). Since Adolph's experiment in 1947, it has been shown in many monogastric animals that when 

diets are diluted with inert materials to produce diets of varied energy concentrations, the animals are able to adjust 

their food intake so that the amount of calorie (and other nutrients) eaten remains constant (Forbes 1986). 

Although this compensation is not perfect, the dietary requirement is more accurately expressed as per digestible 

energy basis rather than per weight of feed (dry matter basis). However, expressing dietary requirements of 

nutrients based on energy content of the diet also involves many problems. The source of energy, whether it is fat, 

protein or carbohydrate, has different effects. Utilization of energy, especially carbohydrat e, differs depending on 

the dietary level and the state of the ingredient (raw, cooked, retrograded). Amino acid composition and their 

bioavailability divert the utilization of protein from growth (deposition) to consumption. Amounts of exercise and 

stresses also divert energy flow from growth to consumption. Heinsbroek (1987) says ME decreas es at increasing 

feeding levels. Tabulating DE or ME values for many feed ingredients is indeed a daunting task, yet the values 

cannot be very accurat e and universal. Expressing nutrient requirements based on per unit body wt/d may be 

acceptable for homeotherms for their normal growth rate/d is known for various age groups. For poikilotherms, 

however, daily growth rate depends so much on rearing conditions (e.g., temperature, stress, feeding rate), which 

makes the use of "per day" expression inappropriate. Dietary requirement of nutrients, particularly for those 

constitute the body, may be most accurately expressed as per growth, which is weight gain, but more precisely lean 

gain or N retention. The protein/ash ratio of the body of animals of the same species is quite constant over the 

different li festages and nutriture except under starvation and malnutrition (Bondi 1987). This indicates that 

expressing dietary requirements of the ash components including P based on the retention of protein (N) should be 

quite accurate, the concept of which may be supported by Rudman et al. (1975), as mentioned above, in that N, P, K, 

Na, and Cl are retained in the body at a fixed ratio at all levels of N intake. Sherman (1920) wrote based on his 

P-requirement studies, "We are probably justified in concluding that about one-fortieth to one-fi ftieth as much P as 

of protein is required in the maintenance metabolism of man." This account, however, may be better suited during 

growth than maintenance. Expressing P requirement relative to N-retention, however, has a practical problem since 

N retention or lean-growth can not be accurately predi cted from feed composition. However, when reporting the 

result of a requirement study, basing the requirement value per N-gain provides a rational basis of comparison with 

the data from other experiments that might be conducted with different diets, fish, and under different conditions. 


20

Practically, the dietary requirement, that has been traditionally reported as per wt of a diet, should be divided by the 

feed effi ciency (gain/diet) of that particular diet, which gives the requirement value as per wt gain. This is the 

dietary requirement value when feed efficiency is 1, which was referred to as Standardized requirement or 

Requirement coeffi cient (Sugiura et al. 2000). The requirement coefficient is universal, and can be multiplied by 

the feed effi ciency of any diet that is to be used in practical feeding. Thus, the dietary requirement for any practical 

feeds (g/g diet) = Requirement coeffi cient (const.) × Feed efficiency of the diet. This simple procedure makes the 

requirem ent value accurate and practical. 

Formulating Low-P diets 

In 1841, Claude Bernard conducted several feeding experiments (under Magendie who was then the chair of the 

Gelatin Commission) to study nutritive value of gelatin. In one experiment, Bernard fed dogs with mutton bones, 

from which P had been removed with hydrochloric acid. Lehmann (1851) wrote, "The ash of the 

protein-compounds consists for the most part of phosphate of lime; Berzelius found 1.8% in the albumen from the 

serum of ox-blood, while Mulder found 2.03% and Marchand from 2.1 to 2.5% in that of the egg; in soluble 

albumen precipitated by great dilution and neutralisation, I found 1.3% of phosphate of lime; in well-washed fibrin 

from the venous blood of a man, I found only 0.694%. Casein, globulin, chondrin, and glutin also contain 

phosphate of lime as an integral constituent. Casein, according to Mulder contains 6% of phosphate of lime, which, 

when the casein is coagulated, is precipitated with it, even when there is a sufficient quantity of free acid in the fluid 

(p. 415-416)." C.Voit (1874) conducted balance trials using ossein, the organic material of bone that remained 

after cooking the bone with hydrochloric acid. Forster (1873) restri cted dogs to nearly ash-free diets. He used 

the residue from the manufacture of Liebig's extract of beef as the source of protein in the diets. It was muscle 

substance from which all water-soluble substances had been extract ed (it still contained ca. 0.8% of ash-forming 

constituents). The animals collapsed in a shorter period than when they were subjected to complete starvation. 

Hoesslin (1882) fed dogs with diets composed of egg white, starch, lard and a salt mixture to study the value of iron 

in blood regeneration. Kolpakcha (1888) determined P 2O 5/N ratio in meat, gelatin, whites of eggs, and yolks of 

eggs. The ratio was much higher in whites of eggs than meat and yolks of eggs. The author reported that gelatin 

was free of P 2O 5. They conducted a series of experiments in which dogs were fed one of these protein sources and 

lard, while in other experiments they were fasted or fed different quantities of meal with same P 2O 5/N ratios or they 

were also fed a diet free of N (starch replaced N sources). The author thought that by measuring P 2O 5/N ratios in 

urine, food and during fast, it can be known whether the N in the urine came from the breaking down of the protein 

of the food consumed or from the breaking down of tissue protein. During fast or when fed N-free food (lard + 

starch), P 2O 5:N ratio in urine became about 1:4 which was the ratio equivalent to that of tissue protein. During 

feeding on meat ration, regardless of the amount of food consum ed the ratio was about 1:7.3 in both food and urine, 

which convinced the author that little protein of tissue is broken down while feeding on protein ration. In passing 

from the meat to the gelatin ration the quantity of N in the urine increased, while P2O5decreased though it did not 

entirely disappear. The author suggested the consumption of gelatin alone cannot prevent the breakdown of protein 

tissue; that is, the organism lives not only at the expense of the gelatin, but also at the expense of its own tissue. 

The author also noted that on the gelatin ration alkaliearth phosphates increased in urine compared with the alkali 

phosphates. "Knowing the relative amount of phosphoric acid to N in the food, tissue protein and urine, the 

quantity of each sort of protein which is broken down may be expressed mathematically." Jordan, Hart & Patten 

(1906) and Hart et al. (1909) prepared a diet very low in P using washed wheat bran, wheat gluten, rice, etc. The 

authors wrote "Phytin can be almost completely removed from wheat bran by mere washing with water, but more 

easily by soaking the bran overnight in warm water to induce a slight acid ferm entation followed by leaching with 

water." The washed bran contained 0.10-0.15%total P (dry basis) compared with 1.4-1.7% in the whole 

(unwashed) bran. Lipschutz (1909) made a diet poor in P using egg albumin, rice, sugar, fat and salt mixture. 

He noted that the excretion of P in the urine of the dog given low-P diet was very low compared with that of P 

supplemented diet. Heubner (1909) reported that rickets could be produced in dogs by feeding diets very low in P. 

He also used egg-albumin as a source of protein. Osborne & Mendel (1918) showed that the lack of suffici ent P 

in diets resulted in a prompt cessation or restriction of growth in rats. They used edestin for the P-free diet and 

casein for the low-P diet to furnish protein in the diets. Addition of inorganic P to the diets brought prompt growth 

responses. Sherman & Pappenheimer (1921) fed wheat flour with no protein sources to produce rickets in rats. 

McCollum (1923) report ed the composition of a diet that produced the most extreme degree of rickets in young rats 

as follows; wheat (whole) 33%, maize (whole) 33%, gelatin 15%, wheat gluten 15%, NaCl 1%, and CaCO 3 3%. 

Day & McCollum (1939) prepared a diet "extremely low in P". The percentage composition of the diet was as 

follows; edestin 16.0, gelatin 4.0, sucrose 61.2, P-free salt mixture 4.0, hydrogenated fat 10.0, and low-P vitamins. 


21

The diet contained 0.017% P and 0.4% Ca. The edestin (technical grade) contained ca. 0.09% P. Jones (1938, 

1939) conducted res earch aiming at developing low-P diet. He wrote, "The greatest difficulty in the preparation of 

a purifi ed diet low in P has been to obtain readily a satisfactory and inexpensive protein. Casein, which has been 

used so widely in synthetic diets, is not suitable as it contains too much phosphorus." and "Crude beef-fibrin at the 

only source of protein in a synthetic diet supports growth at a level comparable to that of casein." The diet he used 

contained only 0.02% P. Phillips et al. (1958) put a small amount of 32 PO 4, varied amounts of Na 2HPO 4 and CaCl 2, 

a red food dye, and cellulose powder (filler) in a gelatin capsule, and force-fed this "synthetic diet" to previously 

starved fingerling brook trout. Later, Phillips et al. (1960) replaced 50% of the cellulose powder in the diet with 

glucose. Ogino & Takeda (1976, 1978) used egg albumin as a protein source in diets to determine P requirements 

for carp and rainbow trout. Lovell (1978) used bovine fibrin (forti fied with valine) and Wilson et al. (1982) used 

egg albumin as the protein source to formulate low-P basal diets in their P requirement studies of cat fish. Shearer 

et al. (1993) compared nutritive values of egg white, blood fibrin, casein, fish meal and one commercial diet based 

on proximate, mineral and amino acid analyses of the ingredients, diets, and the fish (Atlantic salmon, initial wt. 3.6 

g) fed the diets for 8 wk. The growth and feed efficiency of the fish fed blood fibrin and egg white diets, especially 

the latter, were very poor compared with those of fish fed cas ein diet, fish meal diet or commercial diet. They 

reported that P contents of casein, blood fibrin, and egg albumin were all similar, which is questionable. 

McCay’s first aging experiment 

Growth velocity also appears to be critical for nutrients other than those constitute body or skeletal systems. In 

1927, Bing and McCay conducted what became known to be the first modern fish nutrition experiment (McCay et 

al. 1927). The word "modern" implies that the experiment was well-controlled using diets of nutritionally defined 

compositions. Previous to that date, of course, numerous workers conducted "feeding trials" that compared 

different practical-type diets or feed materials of unknown compositions based primarily on economical interest. 

In those trials, a high mortality rate, for example, was described as avitaminosis with no scientific basis. In the 

experiment of Bing and McCay, brook trout fed on low-protein diet had stunted growth, but they lived longer than 

the fast-growing trout fed on high-protein diet. Based on this, they hypothesized, "They behave as if there is 

something in them which is gradually consumed during growth, so that if animals are kept from growing, they live 

longer . . . aging is a process of drying up." 

Murakami's experiments on fish rickets 

Murakami's pioneer research on fish rickets was published only in Japanese as annual reports to the Hiroshima 

prefecture. Ogino (1980) wrote, "Murakami was the first who demonstrated the importance of dietary phosphates 

in fish feeds." This statement too was made only in Japanese. Thus, Murakami's pioneer research has received 

little attention overseas. In 1967-68, Murakami reported cranial deformity and other skeletal abnormalities of 

juvenile common carp. He diagnosed the deformed fish based on careful observations, and without any laboratory 

analysis. The diagnosis included such insightful descriptions as "thin, brittle cranial bones" and "high fatness of 

the fish". He showed that the incidence of deformed fish was higher among the fast-growing than the 

slow-growing group within a population, and the deformity was mani fested only when fish were fed artifi cial diets 

intensively. Small fish (initial size) were more vulnerable. Various possibilities on the cause of the deformity 

such as heredity (genetic effect ), chemical toxicity, hypoxia, starvation, water quality, and parasites were also 

studied, and eliminated by preliminary experiments. The incidence of deformity was higher 1) when feeds were 

offered on a tray suspended in water column (than when feeds were offered by hands) and 2) when fed to satiation 

(than when fed a limited amount) and 3) when reared in flow-through tanks (than when reared in a stagnant system). 

When fish were fed artificial feeds, silkworm pupae, or fish meal, more than 50% of fish showed bone deformity. 

Fortifying the diets with vitamins such as vitamin C, D3 and E did not reduce the occurrence of bone deformity (this 

tested the possibility of vitamin C deficiency reported by Kitamura et al. 1965 as a cause of bone malformation in 

trout). However, when CaHPO 4 and/or McCollum's salt mixture No.185 were added to the diet, the number of 

deformed fish in a population markedly decreased. Also, about 30% of the deformed fish were cured by feeding 

the fish with diets fortifi ed with CaHPO 4 (5%) and McCollum salts (5%), whereas seriously deformed fish did not 

completely return to the normal shape. The author concluded that the bone deformity of fish was due to deficiency 

of P and/or Ca in the diet. It was also pointed out that the contents of Ca and P (total amounts) in the diets did not 

correlate with the incidence of bone deformation. The author speculated that this might be due to different 

absorption efficiency or availability of dietary P and Ca. Murakami (1969) found that dietary supplementation of 

McCollum salts, K 2HPO 4, Ca(H 2PO 4) 2, and CaHPO 4 markedly reduce the number of deformed fish and increas e fish 

growth. Conversely, supplementing the diet with CaCO 3 or Ca 3(PO 4) 2 increased the number of deformed fish, but 


22

did not affect fish growth. The author thought that Ca/P ratio was important and that the number of deformed fish 

was low when Ca/P ratio was 1.5 to 2.0 and high when it was higher than 2.0. The optimum supplementary levels 

were 8% for McCollum salts and 6% for K 2HPO 4 based on fish growth and the number of deformed fish. 

Murakami (1970a) studied effects of various P supplements. Sodium or potassium phosphates were all effective 

to reduce the number of deformed fish and to increase fish growth. Addition of CaCO3 markedly increased the 

number of deformed fish. Supplementing the diet with vitamin C increased the number of deformed fish and 

decreased fish growth. About 40-70% of the deformed fish were cured by feeding a diet supplemented with 

NaH 2PO 4 at 4%. Small fish (0.1 g) required more dietary P than large fish (2 g). The addition of NaH 2PO 4 into 

rearing water also reduced the number of deformed fish. Murakami (1970b) and Hiroshima-tansuishi (1971) 

reported that the supplemental level of NaH 2PO 4 2H 2O for optimum fish growth, feed effi ciency and protein 

efficiency was 4% for a fish meal based diet and a commercial diet. The levels of muscle fat and visceral fat were 

lower, and ash were higher in fish fed the diets forti fied with NaH 2PO 4 2H 2O (2-8%) than in fish fed the unforti fied 

diets. The author suggested the necessity of dietary P for normal energy metabolism. The optimum Ca/P ratio 

differed greatly between fish meal diet and commercial diet, but supplementing the diets with NaH 2PO 4 2H 2O at a 

4% level provided best results regardless of the Ca/P ratio of the diets. The author suggested that this is due to 

different availability of P and Ca in the diets, and that P in fish meal is mainly the bone P and the availability of such 

source (and Ca 3(PO 4) 2) to the fish is very low. Fish fed fish meal diet without supplemental P had higher fat 

content in viscera and muscle than fish fed the diet forti fied with NaH 2PO 4. The protein effi ciency ratio was 

lower in the former fish (39%) than in the latter fish (48%). Dietary P forti fication was also shown to be effective in 

large fish (initial wt. ca. 35 g, final wt. 150-200 g). Tanaka (1971) confirmed Murakami's observation using large 

fish (initial wt. ca 34 g, final wt. 280-400 g). Supplementing a commercial feed with NaH 2PO 4 2H 2O at a 2% level 

markedly increased growth and feed efficiency, and reduced condition factor, body fat and feed cost per gain. 

Murakami (1972) des cribed external signs of P deficiency of carp as deformed cranium, jaw, and pectoral fins, and 

bent caudal peduncle (rhodosis). The author stained Ca in the body of larval-juvenile fish, and noted that the 

deformation was most pronounced in cranium and rib. The author also noted that there was no incidence of 

vertebral curvature (scoliosis). 

P requirement in Carp 

Takamatsu et al. (1975) confirmed Murakami's observations using juvenile carp (initial wt 30-90 g). They 

determined ATPase activity in myosin and actomyosin of skeletal muscles as discussed above. Fish fed high-P diet 

had lower body fat content than those fed low-P diet, but the former fish gained more fat due to their higher growth 

rate. Shitanda & Ukita (1979a, 1979b) and Shitanda et al. (1979) reconfirmed the findings of the former 

researchers. They found that inorganic P levels in blood serum markedly increased after feeding (highest at 1 h 

after feeding) when a fish meal-bas ed diet was supplemented with either Na 2HPO 4, K 2HPO 4 or Ca(H 2PO 4) 2. 

However, the rise of serum P levels was less pronounced when CaHPO4 was used, and there was no increase of 

serum Pi when the diet was supplemented with Ca 3(PO 4) 2. The serum P level was correlated to the amount of 

phosphates supplemented to the diet. Deposition of dietary fat (increase of fat in fish body / dietary fat intake) was 

37-38% in fish fed P-forti fied diet, whereas it was 75-92% in fish fed P-unforti fi ed diet. Hepher & Sandbank 

(1984) found that the growth of common carp was increased by dietary supplementation of dicalcium phosphate or 

monosodium phosphate when fish were fed in tanks with a fish meal-soybean meal based diet or a soybean 

meal-algae meal based diet. When experiments were conducted in earthern ponds in a polyculture system (carp + 

tilapia + silver carp), the growth (harvest) of carp was increas ed only slightly in one experiment, but no effect in 

another by supplementing a commercial diet (control) with monocalcium phosphate or dicalcium phosphate. They 

implied that additional P might be supplied from natural organisms (e.g., phytoplankton, zooplankton, benthos) that 

usually contained P in amounts 1-2% per dry matter. Viola et al. (1986) reported that 0.4% available P was 

adequat e for normal growth of carp weighing 100-500 g. Steffens et al. (1988) fed carp (initial body wt, 200-250 

g) for 84 days at 24ºC. The fish growth was markedly increased when the control diet containing a high level of fish 

meal but no mineral supplement was supplemented with a mineral mixture containing various forms of phosphate 

(water insoluble) at a 4% level. Huang & Liu (1989) reported dietary requirements of P and Ca to be 22-25 mg 

and 33-37 mg, respectively, per 100 g body wt per day for juvenile grass carp for the maximum growth. The 

authors reported that dietary Ca, P, S, Fe, Mg, Co, and Cu greatly affect ed the growth of grass carp fingerlings. 

Takeuchi et al. (1993) estimated the dietary P requirement of juvenile common carp in two 4 wk-feeding trials. In 

the first trial, fish grew from 13 g to 25 g (max) with feed efficiency of 0.98 (max), and in the second one, from 31 g 

to 72 g (max) with feed effi ciency of 1.1 (max). The dietary requirement of available P and DE for minimizing P 

and N excretions and maximizing weight gain and feed efficiency was estimated to be 0.7% and 340-350 kcal/100 g 


23

diet. Schäfer et al. (1995b) fed common carp for 63 days with a soybean meal-fish meal based diet (ca. 0.24% 

available P/DM) with or without supplemental Ca(H 2PO 4) 2. The fish growth and the contents of ash and P in 

dorsal scales and whole body increased in proportion to the amount of P added to the basal diet (ca. 0.42 or 0.60% 

available P/DM). The backbone (defatted) and opercula seems to be less sensitive than scales to dietary available P 

concentrations. Kim et al. (1998) estimated dietary available P requirement for mirror carp to be about 0.7% for 

the maximum growth and minimum loss of P (per kg wt gain). The feed conversion of the diets was about 1.0 

(max). Fish (body wt, initial 18 g; final max 44 g) were fed diets containing fish meal, soybean meal, wheat flour, 

etc. with varied levels of P supplied as Ca(H 2PO 4) 2. The authors calculated the available P content of the 

P-supplemented diets assuming that the P in Ca(H 2PO 4) 2 was 90% available. The final body P content of the fish 

was similar among treatments, but wt gain of the fish clearly responded to the dietary P concentrations. The initial 

fish had even lower concent ration of P in the body than fish fed the diet of the lowest P content (0.24% available P) 

for 8 weeks. This suggests that the dietary requirement might be overestimated since P-deficient fish require P not 

only for growth but also for replenishing the body P pool. The retetion plateau of P has been shown to be 

influenced by the P status of the body or previous diet history (Edwards & Gillis 1959, Sugiura et al. 2000). 

P requirement in Salmonids 

Ketola (1975) studied the dietary requirement of P for Atlantic salmon based on weight gain, feed conversion and 

bone ash content. Incremental amounts of P were added to the basal diet containing soybean meal (70%) and 

CaCO 3 (3%). Fish grew from 6.5 g to only 9.8 g (max.) during a 5 wk feeding period with feed conversion ranging 

1.69-2.75. Although the basal diet contained 0.7% total P, the content of available P might be substantially low 

(not measured). The Ca added to the basal diet could precipitate phytate-P and some Pi. Ogino & Takeda (1978) 

estimated dietary P requirement for rainbow trout in a 6 wk feeding trial using egg albumin-based diets of four P 

levels and two Ca levels. Fish consuming P-adequate diets increased their initial weight about 3 times (initial wt. 

1.2 g, final max wt. 3.6 g) with the feed efficiency of 1.1 (max). The dietary requirement of availabl e P for the 

normal growth was between 0.32 and 0.64% in the low-Ca diet, and more than 1.1% (no plateau) in the high-Ca diet 

(the authors concluded di fferently). Levels of Ca and P in the whole body of fish fed the low-Ca diet increas ed 

proportionately to the dietary P levels (no plateau); however, the ash level reached the plateau at 0.64% P in the diet. 

Among fish fed high-Ca diets, there seemed to be no plateau at all for ash, Ca and P contents in the body. The P 

availability (absorption) in the low-Ca and high-Ca diets was not measured. Watanabe et al. (1980) determined 

the dietary P requirement for chum salmon (initial wt. 1.5 g, final max. wt. 4.9 g) in a 7 wk-feeding trial. Fish were 

fed four times daily to apparent satiation. Feed effi ciency ranged from 33% (lowest P) to 101% (adequate P). 

The diets contained ~0.37% Ca supplied primarily as Ca lactate. The absorption (availability) of dietary P was not 

measured. The estimated dietary P requirement for growth and bone development was 0.5-0.6% as total P. Fish 

receiving the diet of the lowest P content had markedly lower fat content in the body and viscera, which is an 

opposite observation from others. Ketola (1985) devised a new, low-cost diet that contained only a modest amount 

of P supplied mainly from defluorinated rock phosphate. He claimed that the diet supported "significantly slower, 

but adequate growth" and reduced P pollution by more than 50%. The P pollution that the author meant was the 

soluble P that was not retained by the fish. This soluble fraction of P excreted by fish generally represents dietary 

available P that was absorbed by the fish and excreted subsequently via urine due to an excess intake. Apparently, 

the total P in diets was more than the dietary requirement since the basal diet contained soybean meal and casein (or 

corn gluten meal) as the major ingredients, and this diet was further supplied with either dicalcium phosphate or 

defluorinated rock phosphate in the amount about the dietary requirement for the fish. Depending on the 

availability of P in these P sources, the fish simply excrete an excess portion as soluble P. This excess portion must 

be reduced by reducing the amount of available (excess) P in the diet to the minimum requirement level for the fish. 

Unfortunately, the author reported neither the amount of P retained by the fish (body P content) nor the amount 

excreted into feces (P availability), nor even the total amount of P in the diets. Vielma & Lall (1998a) fed Atlantic 

salmon (initial wt 15 g) to satiation for 16 wk. The basal diet containing 4 mg P/g (0.15 mg available P per KJ DE) 

was supplemented with eight graded levels of Ca(H 2PO 4) 2·H 2O. The fish required 0.28 mg available P per KJ DE. 

Increasing dietary P increased P and Ca levels in plasma and bone, whereas liver cholecalci ferol level decreased. 

In P-defi cient fish, the urine P concentration was 0.10 mmol/L before feeding and 0.25 mmol/L after feeding, 

whereas in P-replete fish these concentrations were 1.09 and 5.11 mmol/L, respectively. The apparent absorption 

of P was lower in P-replete fish than in P-deficient fish. Vielma et al. (2002) reported significantly lower tolerance 

of whitefish against high water temperature in dietary P restriction. But, there was no detectable difference in 

low-oxygen tolerance in dietary P deficiency. 


24

P requirement in Catfish 

Andrews et al. (1973) reported that the dietary available P requirement for optimal growth, feed effi ciency, bone ash 

and hematochrit levels in channel cat fish was about 0.8%. Available P content of the basal diet (practical -type) 

was calculat ed based on P availability values of each feed component determined for the chicken. Dove et al. 

(1976) fed channel cat fish in ponds from May until September in 1970 with practical diets low, med, and high in P 

(also Ca) contents, and measured Ca, P, Mg, Na, K, and N contents in the fish body or bones monthly. Fish fed the 

low-P diet had lower P and Ca in the body and, to a lesser difference, in the bone than those fed the high-P diet. 

The difference was small, however, in the first two months of the feeding period. Lovell (1978) estimated the 

minimum dietary P requirement of channel cat fish (initial wt 1.5 g; final max. wt. ca. 5.3) using semi-purified diets 

containing 0.75% Ca with 8 graded levels of P supplied from NaH2PO4, which was estimated to be 90% available to 

fish (measured in a separat e trial). The estimated dietary P requirement for optimum growth and bone 

mineralization was 0.45% as total P. Ca/P ratio did not have a significant effect on weight gain of the fish. 

Wilson et al. (1982) reexamined the minimum dietary P requirement of channel cat fish (initial wt ca. 6 g; final max. 

wt. ca. 49 g). Their estimation for the minimum dietary requirement of available P was 0.33% for growth and bone 

mineralization. The basal diets contained (supplemented) 0.5 and 0.75% Ca. These workers determined the 

apparent P absorption using chromic oxide (for the basal diet). Firdaus & Jafri (1996) fed freshwat er cat fish, 

Heteropneustes fossilis, with casein-gelatin diets of graded P levels (0.06, 0.35, 0.5, and 0.7%) for 6 weeks. The 

fish grew from 12 g up to 22 g (max.). The fish growth increased and feed conversion decreased linearly as the 

dietary P increased. The feed conversiton was 2.11 when dietary P was 0.7%. The haematocrit value, serum P and 

serum Ca increased and the erythrocyte sedimentation rate decreased linearly as dietary P level increased. 

P requirement in Tilapia 

Viola et al. (1986b) conducted three feeding experiments with male tilapia hybrids (initial body wt. 121-275g). 

The initial sizes were large, feeding durations (35-44 days) were relatively short, and thus fish gained the weight 

only 42-65% (max.) of the initial. Differences in weight gain were apparently insignificant, and there were 

essentially no differences in ash, Ca, and P content of the fish. It is diffi cult to draw a firm conclusion based on the 

data presented. The authors estimated available P requirement for tilapia to be 0.45-0.6% assuming that P in fish 

meal and dicalcium phosphate were 70% and that in plant sources were 33% available to the fish. Feed efficiency 

of the diets ranged 45-80%. Robinson et al. (1987) did not see any clear effects of dietary P supplementation in a 

12 wk feeding trial with tilapia, Oreochromis aureus (initial wt. 1.5 g, final wt 11 g max). The basal diet contained 

0.2% P supplied from cas ein and 0.8% Ca, and the feed efficiency of the diet was 40% (max). This suggests that 

the basal diet might contain about enough P. The authors concluded that 0.3% total P was adequate for good 

weight gain and feed efficiency, and that 0.5% total P was required for norm al bone mineralization. 

P requirement in Other fishes 

Davis & Robinson (1987) studied dietary P requirement of red drum. In a 11-week feeding trial, fish increased 

weight more than 10 times (initial 1.2 g; final 14-17 g). Fish fed the basal diet (0.26% total P) had comparable 

weight gain and feed conversion to those fed P-supplemented diets. However, concentrations of ash, P, Ca, and Mg 

in the bones, scales and/or skin were lower in fish fed P-deficient than P-adequat e diets. Scales were more 

sensitive than skin to dietary P levels. The weight of scales (per fish wt) was also lower in fish fed P-deficient than 

P-adequate diets. The P requirement for the maximum bone mineralization was estimated to be 0.86% as total P 

(available P was not measured). Brown et al. (1993) reported the dietary available P requirement of juvenile 

sunshine bass (body wt, initial 2.7 g; final 11-22 g) to be 0.41% for weight gain, 0.46% for feed efficiency, 0.54% 

for maximum bone and scale P concentrations. The basal diet they used contained 0.34% available P. Feed 

efficiency (gain/ feed) ranged from 48% to 62%. These data suggests that the basal diet apparently contained P in 

the amount slightly over the minimum requirement. P and Ca contents of the whole body, serum, bone and scale of 

the fish were similar among treatments, but the weight gain of the fish fed diet of the lowest P was only about a half 

or less than that of fish fed diets of higher P content. The authors (also Baeverfjord et al. 1998) reported an 

incidence of tetany with some mortality of the fish fed the diet of the lowest P. In mammals, tetany is a 

characteristic sign of hypocalcemia often associated with P-overload (e.g., McCollum et al. 1939, pp.372; Harrison 

& Harrison 1979; Hruska & Connolly 1996). Dougall et al. (1996) conducted three experiments to establish 

dietary P requirements for striped bass. Ca and P levels in scales, vertebra, dorsal fin and serum, and weight gain 


25

and feed effi ciency were used as respons e criteria. In the first trial, large fish (initial wt. 321 g) were fed for 14 

weeks but they gained only ca.30%. Consequently, the differences among treatments were very small. In the 

second trial, fish (initial wt 7.9 g) doubled their weight after 6 weeks of feeding with diets of varied P levels (5 

levels ranging 0.15-0.95%P). In the 3rd trial, fish grew only from 48 g to 61-72 g after 10 weeks of feeding, and 

the responces of fish to dietary P levels (5 levels ranging 0.30-0.62%P) were no clear. The authors determined the 

P requirements in the 2nd and 3rd trials based on each of the foregoing respons e criteria, and took an average. The 

averaged dietary P requirement for optimum growth, feed effici ency and serum P level was 0.29%, whereas that for 

ash, Ca, P in scales, vertebra, and dorsal fin was 0.58% as total P (available P was not measured). They also 

reported 15% of fish fed P deficient diet had scoliosis, which could be due to vitamin C defici ency (see below). 

Shim & Ho (1989) tested 3 levels of Ca and 3 levels of P to estimate dietary requirements of Ca and P for guppy, 

Poecilia reticulata. Dietary Ca levels did not affect the growth but P levels affected growth, feed conversion and 

bone ash, Ca and P levels. The authors concluded that the P requirement was between 0.53 and 1.23%. The highest 

feed effi ciency was about 36%, and the fish only doubled the weight after 12 weeks of satiation feeding. The basal 

diet contained 43% of cas ein (P content in casein is notoriously high), but the analytical data showed the diet 

contained only 0.05% P/diet. The authors reported bone deformity such as scoliosis, lordosis and broken-back in 

the group of fish fed diets without supplemental P. The P sources most commonly used in P requirement studies 

are KH 2PO 4 and NaH 2PO 4. Supplementing test diets with such P sources lower dietary pH, which stabilizes 

ascorbic acid in the diets during processing and storage. Thus, low-P diets could likely become defi cient in vitamin 

C, unless the vitamin is stabilized or overforti fied. According to Rose (1938), "Rickets is a disease of the entire 

bone; scurvy affects the growing ends. In rickets, the bone tends to bend; in scurvy, to break". In fish, this is also 

the case; the broken-back syndrome is the sign of vitamin C defici ency (e.g., Lovell 1973), while bending bones are 

the sign of P defici ency (e.g., Shearer & Hardy 1987). Baeverfjord et al. (1998) reported that the bones of 

P-depleted Atlantic salmon were extremely pliable especially opercula and ribs. Scoliosis was observed frequently, 

but there was no incidence of fractures. Shimeno et al. (1994) studied effects of dietary P supplementation on 

perform ance and body compositions of juvenile yellowtail. In a 40-day feeding trial, fish growth, feed effi ciency 

and PER were the highest when fish meal-based diet was supplemented with KH 2PO 4 at a 1.5% level (0.34% as P). 

The dietary P level, however, had little effect on the whole body ash content. The amount of soluble P in diets 

increas ed as the supplemental level of KH 2PO 4 increased. Thus, the percentages of water-soluble P per total P 

were more than 50% in diets high in P. However, the apparent digestibility of P in such diets was only about 

13-33%. El-Zibdeh et al (1995a) estimated the dietary P requirement of redlip mullet Liza hematochiela. Fish 

grew from 3.8 to 44 g (max) in a 14-wk feeding trial and from 27 to 154 g (max) in another trial lasted for 12 weeks. 

Dietary P (total P) requirement estimated based on the maximum weight gain was between 0.37 and 0.54% in the 

first trial and between 0.56 and 0.71% in the second trial. Feed efficiencies were about 91% and 61% in the first 

and second trials, respectively. The authors made numerous other measurements including feed intake, 

hepatosomatic index, condition factor, blood analyses (hematocrit, hemoglobin, total protein, triglycerides, total 

cholesterol, Ca, P), vertebral analyses (lipids, ash, Ca, P, Cu, Zn, Mn, Mg, Fe), and liver analyses (moisture, lipids, 

protein, ash). Interpretations of the data are, however, diffi cult. For example, P content in vertebrae increased 

proportionally to the dietary P intake (no plateau) in the first trial. In the second trial, fish consumed a diet of the 

lowest P had the highest P content in both serum and vertebrae. El-Zibdeh et al (1995b) studied dietary P 

requirem ent for yellow croaker Nibea albi flora. Fish grew from 20 to 66 g (max) in a 14-wk feeding trial with the 

feed effi ciency of about 86% (max). The estimated dietary P requirement based on the weight gain and feed 

efficiency appears to be between 0.42 and 0.65% as total P, while the requirement estimate based on the blood serum 

P is between 0.32 and 0.42% total P (authors interpreted the data otherwise). Elangovan & Shim (1998) reported 

that dietary (total) P requirement for the maximum weight gain of juvenile tiger barb was 0.52% based on the broken 

line analysis of the data. The diets had a feed efficiency of 0.55 (max.). The sources of dietary P were cas ein (basal 

ingredient) and KH 2PO 4 (graded). Chavez-Sanchez et al. (2000) reported that dietary P requirement for an 

American ci chlid, Cichlasoma urophthalmus, was 1.5g/kg diet (sic) for optimum growth. Dietary P levels 

inversely correlated to the carcass fat content of the fish. The authors claimed that optimum Ca/P ratio was 1.3 

when P was supplied from casein and KH 2PO 4 and CaCO 3, respectively. The initial mean body wt of the fish was 

only 0.4 g, suggesting that the fish were fed micro-particulate diets. In this case, a leaching loss of P from the diet 

should be considered significant. What the authors fed might be different from what the fish consumed as 

discussed before (cf. Chapter of phospholipids). The effect of supplemental calcium might also be attributed to its 

possible effect on reducing soluble P in the diet by forming less soluble calcium phosphates. Pimentel-Rodrigues 

& Oliva-Teles (2001) fed juvenile sea bream (initial body wt, 5.1g) for 42 days with 7 diets of varied P 

concentrations (0.37-1.5% total P). The feed efficiency of the normal to high-P diets were 0.92-1.02. Fish fed 

low-P diets had lower growth rate, but the body P content did not differ from those consumed high-P diets. Vielma 


26

et al. (2002) reported lower temperature tolerance (against high temperature) of whitefish in dietary P defici ency, 

while no difference in oxygen tolerance (against hypoxia). Borlongan & Satoh (2001) estimated dietary P 

requirem ent of juvenile milkfish (initial b.w. 2.5g; final b.w. ~16g). The feed efficiency (gain/ feed) was ~65% in 

fish fed P-sufficient diets. They estimated the P requirement in their casein-gelatin based diet to be ~0.85%, dry 

basis, based on weight gain. . 

Part 2. Phosphorus Availability & Absorption 

Etiology of Rickets (background) 

Rickets--- also called Rachitis --- is a pediatric disease characterized by the insuffi cient calci fication of bones. It 

was prevalent among children until aft er its etiology was elucidated in the early 20th century. Rickets is now 

known to be caused by vitamin D deficiency, arising from low dietary intake of vitamin D, from low exposure to 

sunlight (UV)--- which biosynthesizes vitamin D in the skin, and from certain genetic disorders and tumors that 

affect renal vitamin D or phosphatonin metabolisms. Rickets can also result from other factors, including low 

dietary intakes of calcium or P, and malabsorption of P in the intestine. Impaired renal P reabsorption can lead to 

phosphaturia associated with abnormal PTH or phosphatonin level. Like land animals, fish also develop rickets. 

Fish rickets has been known for decades. But, the cause of rickets in fish is invariably due to P deficiency. Even 

though the cause and the preventive methods are well known today, the incidence of fish rickets will necessarily 

increas e in the future. How is this possible? In the subsequent sections, we discuss why the risk is increasing, 

and how the epidemics can be detected and prevented. 

Early studies on P nutrition are closely related to the research on rickets. Both the deficiency and the 

reduced availability of dietary P can lead to the incidence of ri ckets. But, rickets can also result from other factors 

such as the lack of vitamin D, sun light, and calcium, during growth. In the 19th century and the first decade of the 

20th century, rickets was very common in Europe and Northern America. The incidence of rickets was especially 

high in slums and large cities where industrial smoke limited the children to be exposed to sunshine. Osteomalacia, 

the adult form of ri ckets, was also common in many northern countries. In early studies on rickets, several theories 

for its etiology were common: a dietary disorder; poor hygienic conditions; lack of sunlight; poor ventilation; 

prolonged indoor confinement of infants in winter; toxic industrial smoke in large cities; and the lack of exercis es. 

Daniel Whistler (1645) mentioned in his doctoral thesis that the rickets was endemic in England, but unknown to 

the ancients. Whistler said, "Indeed the whole osseous struture is flexible like very moist wax . . ." He also 

considered the disease to be a defect of nutrition, "the lack of the nutritive juice required for the bones and external 

parts. . ." Francis Glisson (1650) gave more comprehensive des criptions of rickets. Glission thought that cold 

and moist air was an etiological factor, rickets itself being a cold and moist disease. Whistler gave a small 

collection of remidies, while Glisson gave a large number of them that comprised one-thirds of the book. Most of 

their suggestions are, however, irrelevant or invalid to the present day knowledge. Whistler suggested exposing the 

abdomen to the sun or covering it with hot sand (to keep the abdomen warm). Glission advised not to eat fish (thus 

unwittingly deprived the source of vitamin D). Underwood (1818) wrote, "It (rachitis) was first noticed in the 

western parts of England, about the year 1628, and is said to have taken place upon the increase of manufactures, 

when people left the villages and husbandry, to settle in large manufacturing towns; where they wanted that exercise 

and pure air, which they had enjoyed in their former situation and employments." He suggested a number of 

interesting remedial procedures, "If the child be too young to exercise itself by walking and such like, . . . She (the 

nurse) has only to dash a few drops of wat er suddenly in its face several times a-day, in the manner often done to 

recover people from a swoon, though less violently. This will oblige the infant to put almost every muscle into 

action . . ." He also recommended, "It has been before remarked, that under every plan, a good diet, air, and 

exercise, especially riding on horseback, are of the utmost consequence; which, if duly persevered in, and the state 

of the stomach and bowels properly attended to, will often effect wonders." Lawrence (1829-30) also wrote, "The 

chief caus e of rickets is a defect in the natural organisation of the individual." The treatment he recommended 

were are, "A good diet judiciously regulated, residence in pure air, attention to clothing--- particularly warm clothing 

in the cold season of the year, and the various means by which an active state of cutaneous circulation can be kept 

up, warm bathing, tepid bathing, sponging the body with warm or tepid water, and, when the individual becomes 

stronger, cold bathing, especially sea-bathing . . . The medical part of the treatment is, perhaps, of less importance." 

There was is no "cod-liver oil" or a similar things in his list of recommendations. Bishop (1848) mentioned about 


27

the causes of rickets, ". . . it most frequently occurs among persons living in low, dark, damp, filthy cellars, and 

ill-ventilated and over-crowded dwellings, such as may be found in many parts of this metropolis, where they are 

not only ill-fed and ill-clothed, but are also denied the enjoyment of a due supply of the great physical agents of 

life,--- namely, light, heat, pure air, and water." Evanson & Maunsell (1847) wrote similarly alike; but they added, 

". . . any thing, in fact, which prevents a healthy nutrition, may produce rickets." They did not mention specific 

nutrients or foods. 

Rickets: 1. Non-nutritional line 

Coote (1869) wrote, "(In rickets) the tissues destined to form the hard bony skeleton remain soft in consequence of 

insuffici ent impregnation or deposit of phosphate of lime, . . ." He suggested the following treatments for rickets, 

"Of all measures the most important is the removal of the child to some country district where the air is pure and the 

soil dry: in most cases the sea-side is preferable. The patient, lightly yet warmly clad, should pass nearly the entire 

day out of doors, and for the moment books of instruction should be disregarded." Palm (1890), an English 

physician who practiced in Japan for several years, noticed that rickets were abs ent in all classes of the native 

population in Japan as compared with its lamentable frequency among the poor children of the large centres of 

population in England and Scotland. He suggested that sunlight should be regarded as a therapeutic agent. 

Findlay (1908) of the University of Glasgow produced rickets in a group of puppies fed on a milk and porridge diet. 

When another group was allowed to run in the open, they did not develop rickets. He, therefore, thought that 

exercise was the explanation of the difference. Raczynski (1912) performed a definite experiment by exposing 

two rachitic puppies to either sunlight or shade for six weeks, and showed that the sunlight-exposed animal had a 

1.5-fold higher bone mineral content. Huldschinsky (1919) reported that children could be cured of rickets by 

exposing their skins to UV radiation, which was subsequently confirmed by Hess, Unger & Pappenheimer (1921) 

in New York City and by Shipley, Park, Powers, McCollum & Simmonds (1921) in Baltimore. It should be 

noted that Mellanby (1919)-- but not Mellanby (1918)-- reported that suet could be one of the most potent 

antirachitic substances along with cod-liver oil and butter. Kramer & Howland (1922) studied normal and 

ricketic rats, and noted that when the inorganic P of the serum was low it could be increased by (1) a few days of 

starvation, (2) by addition of inorganic P to the diet, (3) by addition of cod liver oil and (4) by exposure of the 

animals to UV radiations. Hess & Weinstock (1922) excised a small portion of skin, irradiated it with UV light, 

and then fed it to groups of rachitic rats. The irradiated skin could provide an absolute protection against rickets, 

whereas the unirradiated skin provided no protection. Goldblatt & Soames (1923) identified that when a 

precursor of vitamin D in the skin (7-dehydrocholesterol) was irradiat ed with sunlight or ultraviolet light, a 

substance equivalent to the fat-soluble vitamin was produced. Hess (1924) discovered that irradiating a 

rickets-producing diet with UV light conferred on the diet anti-ricketic properties. Steenbock & Black (1924) also 

showed that vegetable oils, devoid of fat-soluble vitamins, acquired the calcifying properties of vitamin D when 

exposed to UV radiation. 

Rickets: 2. Nutritional line 

The history of cod-liver oil use as a medicine has been discussed in depth by Guy (1923) and Hess (1929). 

Cod-liver oil appeared to be a folk medicine of Manchest er where the weather was frequently rainy and sunlight 

seldom fell on the ground. In 1789, Percival of Manchester stated that cod-liver oil had been dispensed so largely 

in the hospital here. He spoke about a letter of Dr. Darbey written in 1782 reporting the great effect of cod-liver oil 

for the remedy of chronic rheumatism. Bennett (1848) said that Dr. Bardsley had written in a medical report of the 

Manchester Infirmary in 1807 about the efficacy of cod liver oil for osteomalaci a. Schenck (1826) published a 

paper on the remarkable value of cod-liver oil for curing rickets of children. However, until the latter half of the 

19th century, major articles on rickets did not state anything about the use of cod-liver oil. Jenner (1860) wrote, 

"Cod-liver oil is considered by some French writers of repute a speci fic in rickets. . . . But although my experience 

of cod-liver oil does not confirm the statements of Bouchut, it enables me to say that it is a very valuable remedy." 

He said, "rickets causes, primarily or secondarily, more deaths than any other disease of childhood." He added, 

however, "There is no specific for the cure of rickets." He thought cancer and rickets were both nutritional diseases. 

He also wrote, "It is not probable that there is any lack of lime in the blood, seeing that one secretion from the blood, 

viz. the urine, was found, in Marchands's experiments, to contain six times its normal quantity of lime-salts." Also, 

"The health of the mother, however, has a decided influence on the development of rickets in the child.", and ". . . 

the child must be frequently taken out of door. . . it should be removed into the country. Dry, bracing sea air is the 


28

est." He also recommended numerous other remedies, most of which were, however, questionable. The gravity 

of rickets was evident in those days as Jenner also wrote, "Can we wonder that rickets is prevalent among the poor 

of London? Can we fail to wonder that geography, history, and crochet-work form so large items in the instruction 

imparted at our national schools, and the doctrines of life so small. Let the girls there educated be taught that 

Constantinople is the capital of Turkey if it be any advantage for them to know it, but let them also learn how to 

dress, nurse, feed, and lodge an infant, so that it may run a fair chance of not swelling the amount of that truly awful 

column in the Registrar-General's returns--- Deaths under one year." Dick (1863) fed dogs with meat, bread and 

broth, and experimentally produced rickets. This convinced him to say that rickets is induced by improper or 

insuffici ent food, especi ally with bad milk. He wrote (in the footnote!), "About three months after the above was 

written, the dogs completely recovered under the administration of cod-liver oil, with a bread-and-milk diet. Only 

their fore-legs remain slightly bent." Brodhurst (1868) gave numerous recommendations for the treatment of 

rickets, including cod-liver oil as well as warm clothing, a diet composed mainly of animal sources, dry and pure air, 

tepid bathing, various preparations of iron, and lastly nitro-muriatic acid bath (which, he said, was a recourse and of 

great value when used occasionally). However, the author was obviously uncertain about the value of cod-liver oil. 

Coote (1869) recommended the diet that was light, nutritious, consisting chiefly of milk and farinaceous food, of 

fruit and vegetables, and meat should be given sparingly. He objected repeatedly to the use of cod-liver oil since 

the stomach seemed to reject it. He said that the same benefits could be obtained by regulation of the suitable 

articles of diet. "When the bowels are out of order", he said, "the alimentary canal should be cleared by the use of 

gentle pugatives, such as rhubarb and magnesia, or rhubarb and gray powder (i.e. mercury), or a dose of castor oil." 

He also recommended the use of iron preparations, chalybeate waters, quinine, etc. Interestingly, only in the 

previous year in the same journal, did Gee (1868) strongly recommend the use of cod-liver oil in the treatment of 

rickets as he wrote, "And in cod liver oil we possess a pharmaceutical agent worthy of a place beside iron, Peruvian 

bark, and mercury. We ought to lose no time over the symptoms of rickets; slight catarrh, diarrhoea, paleness, a 

tendency to fits, these will all disappear under cod liver oil: give expectorants, purgatives, styptics, and the rickets 

will increase under our eyes. . ." Gee also mentioned about the dentition in rickets, "The teeth tend to decay-- 

owing to deficient enamel, . . .", and he also presented the cases of delayed dentition among rachitic children. 

Smith (1881) wrote, "The medicines which are of undoubted efficacy in rachitis are cod-liver oil and lime." 

Bland-Sutton (1889) experimentally demonstrated that feeding crushed bone and cod liver oil cured rickets of the 

lion cubs at the Zoological Gardens in London. Hopkins (1906) expressed his belief that scurvy and rickets are 

disorders caused by diets deficient in unidentified trace nutrients, which he called "accessory food factors". 

Schabad (1910) in Petrograd cured rickets of a four-year-old child by adding cod liver oil to a diet of bread and 

milk. He found based on the balance that the absorption of Ca and P markedly increased by cod liver oil. Hess & 

Unger (1917) in New York City also showed the therapeutic effect of cod liver oil by treating ricketic children. 

Edward Mellanby (1918) of Great Britain also demonstrated that rickets is a nutritional disease. He reported that 

certain diets caused rachitic changes in the bones of puppies and that the inclusion of certain substances in the diet 

led to a normal bone development. The substances preventing rickets were meat, butter, cod-liver oil and among 

others, and the substances not preventing rickets were casein, linseed oil, yeast, protein of meat, etc. In this short 

report, he also mentioned, " . . . it seems clear that rickets is a deficiency disease of the type of scurvy and beri-beri. 

Similarly the anti-rachitic accessory factor has charact ers related to the growth accessory factor, although it is not 

identical with the latter, since rickets is rather an abnormality of growth and is most prominently shown in quickly 

growing animals." Mellanby (1919) wrote, ". . . large and rapidly growing children most often suffer from rickets, 

whereas marasmic children generally escape. It is, therefore, diffi cult at first sight to associate a disease of rapid 

growth with a deficiency of fat-soluble A which is, according to accept ed teaching, necess ary for growth." May 

Mellanby (1918) also wrote, "As a general rule, it is difficult to associate rapid growth with ill-health, and yet we 

have seen in these experiments that the teeth of the more rapidly growing puppies are worse . . ." Unfortunately, 

however, he became compromised in his position as Mellanby (1919) also wrote, "Whether the anti-rachitic factor 

is fat-soluble A as previously understood is therefore undecided, but, on the whole, these substances appear to be 

identical." Mellanby (1921) also wrote, "The action of fats in rickets is due to a vitamin or accessory food factor 

which they contain, probably identical with the fat-soluble vitamin.", and “ the substance in fats stimulating the 

calci fication of bones is probably the same as fat-soluble A, that is the factor which stimulates growth in rats.” 

Later, Mellanby (1950) reflected on this, "One of my great intellectual diffi culties with this particular line of 

investigation was that accessory food factors were called 'growth factors' and I observed early on that rickets was a 

disease of growth--- no growth, no rickets. It was diffi cult to believe that a deficiency of something essential for 

growth could be responsible for rickets." McCollum et al. (1922) demonstrated that the factor in cod liver oil, 

which promotes growth and prevents xerophthalmia, differs from that prevents rickets by using oxidized cod liver 

oil. The oxidized oil lost vitamin A activity (c.f. Hopkins 1920 reported this method); however, it still retained 


29

anti-rachitic property. McCollum, therefore, named this factor vitamin D. McCollum's demonstration, however, 

was incomplete for Mellanby. Thus, Mellanby argued that there was no guarantee that the oxidation of cod liver 

oil completely destroyed vitamin A. A trace amount of vitamin A, which could remain in the oxidized oil, might 

not be enough to prevent xerophthalmia, but enough to cure rickets. 

Vitamin D and P utilization 

Research on vitamin D is very diverse. This section presents only a cursory review of vitamin D research relat ed to 

P utilization. A pioneer research on vitamin D can be traced back to Mellanby (1919). But, a substantial number 

of researches or observations on cod liver oil and rickets had been reported much earlier. They were already 

mentioned in the previous chapters. Kramer & Howland (1932) concluded that with minimal amount of vitamin 

D, the Ca and P of the serum of rats varied directly with the Ca and P concentrations in the diet, and that vitamin D 

stabilized the Ca and Pi concentrations in the serum. Hannon et al. (1934) administered a small amount of vitamin 

D 2 over a period of 16 days to a patient suffering osteomalacia. The beneficial effect of this vitamin on Ca and P 

metabolism continued for at least 4 months after administration of the vitamin had ceased. Liu et al. (1940) noted 

that vitamin D supplementation rapidly improved Ca and P retention only when there was no reserve of the vitamin 

in the body. McCance & Widdowson (1942a,b) and Hoff-Jorgensen et al. (1946a,b) found that supplementing a 

diet containing phytate with vitamin D 2 did not increase Ca absorption. On the other hand, some workers 

consistently showed in rats and chicks that dietary vitamin D markedly increased the utilization of phytin-P 

(Krieger & Steenbock 1940, Boutwell et al. 1946, Spitzer et al. 1948, Steenbock et al. 1953). Mellanby (1949) 

also reported that vitamin D lowered the amount of phytate P in the feces of dogs. Sommerville et al. (1985) 

found that lowering dietary P markedly increased plasma 1,25(OH) 2D 3 in the chick, which was further increas ed by 

increasing the level of dietary cholecalci ferol. Edwards (1993) reported dietary 1,25(OH) 2D 3 supplementation 

increas ed phytate-P utilization in chickens. 

McCay et al. (1927) fed brook trout fry for 12 weeks. The growth and mortality of the fish fed a 

casein-starch diet supplemented with vitamin A and D (as cod liver oil) did not differ from those fed the same diet 

but without the supplemental vitamins. Phillips et al. (1952) fed brook trout (initial wt. 0.94 g, wt gain ca.1200%) 

for 20 weeks with a practical-type diet, and did not find any effects of dietary vitamin D3 supplementation on the 

growth, feed efficiency, and proximate body composition. Phillips et al. (1955) also failed to see any positive 

effects of vitamin D on growth, feed efficiency, and mortality in brook trout (initial wt. 1.4 g, final wt. ca.10 g). 

This time, fish were fed for 20 wks with Wolf's synthetic diet (less cod liver oil) supplemented with corn oil with or 

without the vitamin. Phillips et al. (1959) did not see any noticeable effects of dietary vitamin D2 in brook trout 

on the assimilation of 32 P administered in one meal. However, Lovell & Li (1978) demonstrated that vitamin D is 

an essential nutrient for growth and bone calci fi cation in channel catfish fingerlings. The responses reached a 

plateau at 500 IU/kg diet. When a casein-based semi-purifi ed diet low in Ca (0.05%) but adequate in P was 

supplemented with the vitamin, fish growth and the bone mineral contents (ash, P, Ca) increased markedly. With 

adequat e amounts of vitamin D in diet, dietary Ca was apparently unnecessary (water contained 14 ppm Ca). 

When a diet low in P (0.18%) but adequate in Ca was supplemented with the vitamin, fish growth and the bone 

mineral contents did not increase. Barnett et al. (1982a) noted that a casein-based semi-puri fied diet free of 

vitamin D3 did not alter Ca, P, AP, and Mg levels in plasma, and Ca and P levels in the bone of rainbow trout after 

168 days of feeding. However, the fish growth differed markedly, which correl ated to the amount of vitamin D 

added to the diet. Vitamin D3 was found to be more potent than vitamin D2. The feed conversion ratio and body 

fat content decreased as the dietary level of vitamin D increased. There was a dose-dependent occurence of tetany 

(up to 56%) among fish fed diets low in the vitamin. Barnett et al. (1982b) confirmed previous observations. 

Fish fed a vitamin D free diet for 280 days had poor growth (47%) compared with growth of fish fed a diet 

containing vitamin D3 in the amount of 1600 IU/kg. The fish in the vitamin deficient group had a higher incidence 

of tetany (88%) than fish fed vitamin D-supplemented diet (13%). No fish showed vertebral abnorm ality, and 

apparently no motality was recorded associat ed with tetany. The vitamin deficient fish showed muscle weakness, 

but they were neither hypocalcimic nor hyperphosphat emic. The authors therefore suggested that the functions of 

vitamin D in trout appeared to be quite different from those in terrestrial animals. 

Avila et al. (1999) fed rainbow trout (body wt. 56 g) for 7 or 8 days with P-suffici ent diets (0.6%P) differing 

in VD 3 content (0, 300, 2,500, 10,000, 40,000 IU/kg). Plasma Pi was slightly higher (8.3 vs 7.0 mmol/L) in fish 

fed diets containing 2,500-40,000 IU VD3/kg than those fed diets containing 0 or 300 IU VD3/kg. However, 

plasma levels of 25(OH)D 3 and 1,25(OH) 2D 3 did not differ. Increasing the level of dietary VD3 also did not 

increas e an in vitro Pi uptake from the intestine. Also, the iIn vitro Pi uptake did not differ among tissues 

pre-incubated in a solution containing VD 3, 25(OH)D 3, 1,25(OH) 2D 3, or Ringer (placebo). Ashok et al. (1999) fed 


30

freshwater fish Labeo rohita (Rora) with diets containing vitamin D 2 (550, 1,100, 1,650 IU/kg) and vitamin D 3 

(1,100, 1,650 IU/kg). The growth, feed efficiency, mortality, carcass protein, fat, Ca and P contents did not differ 

between vitamin D-defici ent fish and fish fed any forms of the vitamin at any doses. Vielma & Lall (1998b) found 

that hepatic cholecal ci ferol content of Atlantic salmon was 58.5 ng/g when fish were fed for 15 weeks with a 

low-P-low-Ca diet (3.1 g P, 1.3 g Ca /kg), and 9-10 ng/g when they were fed diets supplemented with either Ca or P. 

Vielma et al. (1999) reported that growth and feed effi ciency of rainbow trout (initial wt 12.4 g) were not influenced 

by dietary cholecal ci ferol and P levels in a 20 week feeding trial. Liver cholecalci ferol concentration was higher 

(4.9 vs 9 ng/g) when fish were fed diet containing 2600 IU of cholecalci ferol /kg diet than when they were fed 100 

IU/kg, but dietary P had no effect on the hepatic concentration of the vitamin. A high dietary P level increased 

urinary P concentration, whereas dietary cholecalci ferol level had no effect. In mammals, dietary P restriction 

increas es 1,25(OH)D3 or calcitriol, but in trout, Coloso et al. (2001) did not see any such change based on 

radioimmunoassay. Coloso et al. (2003) reported that 1,25(OH) 2D and 25(OH)D concentrations in plasma of 

rainbow tourt (bw 73 g) did not respond to 31d of dietary P restriction (0.3-0.6% total P in semi-purified diet). 

Mellanby's Toxamin Theory 

May Mellanby (1918) published a paper that contained a section entitled "Harmful nature of modern dietary in 

regard to the teeth", in which she wrote, "Our diet, particularly that of the poor, is now more than ever made up of 

specially prepared cereals, such as wheat, rice, oats, &c. . . . There is no doubt that our modern dietary is harmful as 

far as the teeth are concerned." Edward Mellanby (1918), May Mellanby's husband, first developed standard diets 

that produced rickets on puppies (such diets contained bread or cereals), and then added to the standard diets a test 

substance in order to determine their effect on the development of rickets. Mellanby (1919) wrote, "Since the 

dietetic problem is one of balance, foodstuffs which contain no anti-rachitic factor cannot be considered as neutral, 

but as positively rickets-producing, for the more of them that is eaten the greater is the necessity for foods 

containing the factor. Since there is a limit to what a child can eat, the inference is obvious. It is probable that 

bread is the worst offender, and to allow bread to form too large a part of an infant's dietary seems to me to be 

courting disaster. The same statement may apply to other cereals, but this has not been worked out to any extent." 

Subsequently, Mellanby (1922) showed that rickets-producing power vari ed greatly among the kinds of cereals--- 

oatmeal the worst followed in order by brown flour, barley, rice, and white flour. Mellanby (1925) reported that 

oatmeal and maize had a powerful rachitogenic effect. He wrote, ". . . the amount of Ca and P in the food is of but 

secondary importance in the control of the deposition of these elements in growing bone. In view of the evidence 

of interaction and balance among food constituents provided by this investigation, the value of the expression 

‘optimum Ca content of a diet’, so commonly used nowadays, must be doubted. The optimum varies every time 

the other elements of diet are changed." Mellanby (1926) found that a powerful anticalci fying cereal like oats 

lost some of its action after cert ain simple forms of chemical treatment, namely, 1) boiling with acid, and 2) malting 

followed by heating. This crude experiment showed that cereals contained a definite anticalci fying substance 

whose action could now be modified in two entirely different ways, 1) by adding vitamin D and to a lesser extent by 

adding Ca salts, and 2) by destroying the hypothetical toxic substance by boiling with acid or by malting. 

Mellanby (1926) named this unidentified harm ful substance(s) in cereals 'Toxamins', which was later (1934) 

identified as phytic acid. The toxamin prevented utilization of Ca, and this toxic action was antagonized by the 

anti-rachitic vitamin. In fact, Holst (1927) also wrote, "If oatmeal is extracted with hydrochloric acid and the 

extract is given in addition to the starch, the animals develop rickets. The rickets-producing factor of oatmeal must 

therefore be ascribed to some toxic substance. Evidence is given to show that this substance can pass through 

parchment -paper and that it can be precipitated with alcohol. Rickets produced by feeding with cereals can be 

prevented by the administration of calcium-s alts, whereas phosphates have no such effect." However, back to 

Mellanby (1919) paper, he also wrote, "It was found that adding orange juice did not prevent rickets. Further, that 

the addition of 5g. calcium phosphate, or doubling the separated milk and so increasing the calcium intake in this 

form was without preventive action on the development of the disease." Green & Mellanby (1928) showed that 

oatmeal could be boiled with water or heated in the dry state at 120°C for 18 hours without correction of its 

anticalci fyig properties. If, however, it were boiled with 1% HCl for 1.5 hours and then neutralized, the product 

was found to have lost its anticalci fying effect. 

Steenbock, Black & Thomas (1930) drew attention to the possibility that cereals contain a form of P less 

available to rats than inorganic P. Templin & Steenbock (1933b) showed that the disappearance of rachitogenic 

effect after boiling with acid was accompanied by a change of organic P to inorganic P in the cereal, and suggested 

that the organic P might be less available than inorganic P. Then, Bruce & Callow (1934) showed that phytic acid 

forms an insoluble Ca salt and interferes with the absorption of Ca in rats. Lowe & Steenbock (1936a,b) and 


31

Krieger & Steenbock (1940) confirm ed this observation in rats. McCance & Widdowson (1935, 1942a,b) 

confirmed the same effect in humans. They reported that subjects receiving white bread to which sodium phytate 

was added showed a large negative balance of Ca absorption, and that the absorption of Ca and P from brown bread 

was increas ed when it was dephytinized. They found that 20 to 60% of the phytate in wheat flour was excret ed 

unchanged in the feces. They suggested that the utilization of phytin P was due to bacterial action in the intestines. 

Harrison & Mellanby (1939) showed that commercial phytin (Ca-Mg phytate) is not rachitogenic, while sodium 

phytate or phytic acid prepared from a commercial phytin or oatmeal is definitely rachitogenic. They concluded, 

“. . . phytic acid in a rachitogenic cereal like oatmeal immobilizes almost all of the Ca contained in the cereal by 

converting it into an insoluble, relatively unavailable, Ca phytate and further, that the excess of phytic acid (over and 

above that required to precipitate the Ca of the cereal) can exert an additional anticalcifying effect by precipitating 

other Ca present in the non-cereal part of the diet". Cruickshank et al. (1945), however, noted that 94% of the 

phytate disappeared from the gut. Hoff-Jorgensen et al. (1946a,b) found that an increase of sodium phytate in 

diets for infants largely decreas ed the Ca absorption. Waldroup et al. (1964) showed that the availability for 

chicks of the P of free phytic acid and sodium phytate was far great er than that of cal cium phytate. Currently, it is 

understood that the availability of phytate P decreas es as dietary Ca level increases. The availability of phytic acid 

is variable depending on Ca concentration in the diet. Reciprocally, availability of Ca depends on the concentration 

of phytic acid in the diet. The formation of Ca phytate in the digestive tract has been shown to reduce the 

availability of both. Calcium phosphate, however, seems to be less reactive to phytate than Ca carbonate. 

Sodium phytate is soluble and easily destroyed by intestinal, bacterial or plant phytase; however, Ca-phytate is 

insoluble at neutral pH and the Ca-phytate precipitate is resistant to hydrolysis. More than six decades before 

Mallanby, however, Janner (1860) wrote, "And even the frequency of rickets in London has been supposed to 

depend on adulterations of the bread whereby its lime-salts are deprived of their solubility." Apparently, they 

already had a clear idea on how nutrient interactions are important in determining the availability or absorption of 

nutrients in foods. The availability of P in each food ingredient is, therefore, cannot be a single fixed value, but it 

also depends on other compounds that are consumed at the same time in a single meal. 

Availability of phytate-P 

Jordan, Hart & Patten (1906) wrote, "Trypsin and pepsin had no effect to split the free acid of phytin and its 

simple salts into inorganic forms. The influence of other enzymes, such as erepsin and of bacterial ferments, has 

not been tested but this hardly seems probable to effect a cleavage of phytin. The complete disappearance of 

phytin from the intestinal tract of the cows indicates that phytin was absorbed, metabolized, and excreted through 

the feces in inorganic forms." Hart et al. (1909) expressed a similar belief that phytin was absorbed from the 

intestinal tract of pigs and afterwards undergoes cleavage by a phytin-splitting enzyme, phytase, that had been 

reported previously by the same authors to be present in the blood and liver of mammals (McCollum & Hart 1908). 

However, Starkenstein (1910) showed that phytin was not directly absorbed from the alimentary canal of animals, 

and that the breakdown of phytin as occurred in the intestine was likely the result of bacterial action. Rogozinski 

(1910) working on two dogs and a man separated P in their feces into different groups: lecithin-P, phytin-P, 

inorganic-P and protein-P. The phytin was much more completely absorbed by the human being than by the dog, 

and the feces phytin was not increased in human being by the ingestion of phytin. Human feces contained much 

larger proportion of lecithin P than in dog feces. Steenbock, Black & Thomas (1930) suggested that cereals 

contain a P compound less available to rats than inorganic P. Bruce & Callow (1934) showed that the P of phytin 

was much less available to rats than that of sodium P, and that the relative curative effect of these substances on 

rickets, when given with small quantities of vitamin D, was dependent on this difference in availability. The 

relative unavailability of phytic acid P was also shown in humans by McCance & Widdowson (1935) who found 

that 20 to 60% of the phytin was excreted unchanged in the feces. Lowe & Steenbock (1936b) demonstrated that 

phytin-P is poorly available to the rat in contrast with phosphoric acid and sodium glycerophosphate. Lowe et al. 

(1939) reported the availability of phytin-P to the chick using disodium P as a standard. As mentioned above, the 

availability of phytate-P is dependent on the concentration of Ca that can bind and precipitate phytate-P in the 

intestine as calcium phytate. Phytate-P also reduces trace mineral absorption by its strong chelating property, and 

this effect seems to increase in the presence of free Ca ions (Anon.1967). The following reviews on phytic acid 

are useful and comprehensive (Mellanby 1950, Taylor 1965, Nelson 1967, Cheryan 1980, Reddy et al. 1982, 

Maga 1982, Morris 1986, Cosgrove 1980, Lasztity & Las ztity 1990). 


32

Reducing phytate P by non-enzymatic approaches 

There are numerous non-phytase methods that can reduce phytic acid levels in plant ingredients. I have already 

mentioned about the method of Hart et al. (1909), in the section of low-P diet in Part 1, that phytate can be washed 

off with or without acid-fermentation. Holst (1927) reported that oats contained a rickets-producing factor which 

could be extracted with 0.5% HCl. Green & Mellanby (1928) showed that oatmeal could be boiled with water or 

heated in the dry state at 120C for 18 hours without correction of its anticalcifying properties. If, however, it were 

boiled with 1% HCl for 1.5 hours and then neutralized, the product was found to have lost its anticalcifying effect. 

Templin & Steenbock (1933a) demonstrated that superior calci fi cation was produced by immature yellow dent field 

maize as contrasted with the corresponding mature maize of the same vari ety and grown under identical conditions. 

Lowe & Steenbock (1936b) confirmed this observation. Toma & Tabekhia (1979) reported a large loss of phytate 

in milled rice by cooking. Satoh et al. (1998) also mentioned that the extrusion cooking reduced phytate P in canola 

meal by 10–30%. However, Burel et al. (2000) did not see any noticeable difference between heat-treat ed and 

untreated rapeseed meal in phytates and phytic acid contents based on chemical analyses of the ingredients. In vivo 

digestibility experiment, however, showed clear improvement of P availability (by heat treatment) in both trout 

(availability increased from 26 to 42%) and turbot (from 49 to 65%). In this study, turbot seemed to utilize P in the 

ingredient so much greater than trout. Reddy et al. (1978) did not see any break down of phytate during cooking of 

black bean seeds. Edwards et al. (1999) did not find any effect of steam-pelleting or extrusion-pelleting on the 

utilization of phytate P in a corn-soybean meal diet by the chick. After all, autoclaving at high temperature, that is 

necessary to reduce phytate, also reduces heat-labile amino acids (Rackis 1974; de Boland et al. 1975). The 

microwave heating of full-fat soybean reduces phytic acid content (Hafez et al. 1989). Ologhobo & Fetuga (1984), 

however, observed little effect of cooking (at 15 psi or ~121°C for 15 min and autoclaving at 105°C for 20 min) on 

phytate content of soybean, lima beans, and cowpeas. Han (1988) reported that soybean meal and cottonseed meal 

contained 2.3% and 4.4% phytate, of which 60% and 50%, respectively, were water-soluble and easily removed by 

washing with water for 1 h. Washing soybean meal with 1 N HCl removed 87% of phytate. Intensive reviews of 

this subject are found elsewhere (Maga 1982; Erdman & Poneros-Schneier 1989, Sandberg 1991). Single-gene, 

non-lethal low phytic acid (lpa) mutations in corn and barley cause the seed to store most of P as inorganic P instead 

of as phytate P (Raboy & Gerbasi 1996). Replacing ordinary grains in feeds with low-phytate grains can reduce 

fecal phytate P and total P excretions in chickens (Ertl et al. 1998) and trout (Sugiura et al. 1999). Spencer et al. 

(2000) reported that bioavailability for pigs of P in ordinary and low-phytate corn was 9% and 62%, respectively, 

when determined based on bone-breaking strength and the slope-ratio assay. Raboy (2000) wrote about the 

development of low-phytic acid lines of crops that may have benefits in human and animal nutrition by increasing 

bioavailabilities of trace minerals such as zinc and iron, and P. Reducing phytic acid content in various crops up to 

99% seems to be possible by this approach. 

Reducing phytate P by phytase 

Suzuki et al. (1907), while isolating the anti-beriberi factor in rice bran, discovered an enzyme phytase in the bran 

capabl e to breaking phytin down to inositol and phosphoric acid. They suggested that phytin was inositol 

hexaphosphoric acid. Plimmer (1913) studied the digestion of various P compounds with crude enzymes extracted 

from various sources. He reported that phytic acid was digested readily only by an enzyme in the bran extract. 

Anderson (1915) reported that wheat bran is especially rich in phytase. Bioavailability of phytate P in plant 

ingredients is largely dependent upon the amount of phytase available for the hydrolysis. Soaking or adding water 

to cereals activat es naturally occurring phytase. This procedure is particularly effective for wheat, barley and rye 

due to their high phytase content. Oats, cottonseed meal and rape-seed meal have low phytase activity, while 

soybean meal and corn contain no phytase (Møllgaard 1946). Steeping in acidified water (pH 4.5-5.0) is more 

effective to degrade phytic acid in those cereals than steeping in water (Mellanby 1950, Fredlund et al. 1997). 

Stone et al. (1984) reduced phytate in canola meal by mixing it with wheat bran (a source of phytase) and fish silage (a 

source of acidifier), and incubating this mixture at room temperature. Heating or cooking these cereals inactivates 

inherent phytase and reduces phytate digestibility. Fermentation naturally causes a reduction of pH and creates an 

environment to optimize phytase activity. The intestinal or ruminal bacterial populations are important sources of 

phytase in some animal species. Generally the bioavailability of phytate P is high (more than 50%) in ruminants, 

about 33% in pigs, and less than 10% in the chick (Anon.1984). Edwards & Veltmann (1983), however, found 

relatively high phytate P retention (~39%) by broiler chickens fed a corn-soybean diet. Mohammed et al. (1991) 

reported even higher digestibility of phytate P (50%) in a corn-soybean diet by the chick, and this was further 

increas ed up to 77% by reducing Ca (1/2) and increasing cholecal ci ferol (x 100) contents in the diet. Nelson et al. 


33

(1968) obtained phytases from culture filtrates of different sources of Aspergillus species, and digested phytate in 

soybean meal by this enzyme preparation. The authors compared the effects by the chick bioassay. Cain & Garling 

(1995) conducted a similar but simplified experiment with rainbow trout. Numerous researchers have reported some 

favorable effects of supplemental microbial phytase on the availability of P, trace minerals and other dietary nutrients. 

Rodehutscord & Pfeffer (1995), Riche & Brown (1996), Vielma et al. (1998), Forster et al. (1999), Sugiura et al. 

(2000) used rainbow trout, Jackson et al. (1996), Eya & Lovell (1997), Li & Robinson (1997) used channel catfish, 

van Weerd et al. (1999) used African cat fish, Schäfer et al. (1995) used carp, and Hughes & Soares (1998), 

Papatryphon et al. (1999) used striped bass. The degree of effectiveness of phytase seems to be considerably 

different from one study to another. The differences are apparently due to different basal diets used by the above 

workers rather than to the different species. Rodehutscord & Pfeffer (1995) reported that P availability for 

rainbow trout increased from 25 to 57% when a diet containing 55% soybean meal was supplemented with 1000 

units of microbial phytase per kg diet. The authors suggested that practical relevance of the finding applies only 

to diets in which protein is supplied almost entirely by plant products. In carp, the apparent availability of P 

increas ed from 32% to 49% when a diet containing 53% soybean meal was supplemented with 500units of microbial 

phytase per kg diet (Schäfer et al., 1995). Since microbial phytase is almost inactive at pH 7 or higher; and the pH 

of the gut of agastric fish is about 7 or higher (Maier & Tullis 1984), the enzyme may not be fully effective in carps. 

The effect of phytase may be increased i f it is in a low-Ca diet, which is then weakly acidified. Sato et al. (1997) 

reported that carp fed a diet supplemented with microbial phytase had markedly lower P excretion than those fed the 

unsupplemented diet. However, phytase-supplemented diet contained much less P (less P supplement), and the fish 

fed such diet had signs of P deficiency, including lower ash, Ca and P contents and a higher lipid content in the body, 

although their growth was apparently unaffected during 56 days of restricted feeding. In striped bass, Hughes & 

Soares (1998) reported an increase of P availability by phytase with a plant-based diet, and suggested the level of 

1000units per kg diet was optimal. Storebakken et al. (1998) added commercial phytase to soy protein 

concentrate (ca. 15400 units phytase/kg soy protein concentrate), and incubated at room temperature overnight to 

reduce phytic acid content in the ingredient. This procedure greatly increas ed the solubility of P in the ingredient 

from 7% (untreated material) to 70% (phytase-treated material). The test diets were prepared by an extruder, and 

contained fish meal, soy protein concentrate (phytase-treat ed or untreated), wheat, dicalcium phosphate and other 

ingredients. Fish (Atlantic salmon) were fed in seawat er every hour, 24 hours a day to true satiation (over fed and 

uneaten pellets were collected and counted). When fish are fed in such a manner, leaching of dietary components 

is inevitable while feed pellets were floating or suspending in the water or by washing in the mouth of the fish. 

What the fish ingested and consumed could be di ferent from what the investigators fed as dry pellets. This 

difference may not cause a serious effect in determining digestibility of dietary nutrients since soluble components 

in diets are generally digestible by the fish (except phytate-P). The leaching loss, however, will be a direct source 

of error when calculating nutrient retention based on balance. Oliva-Teles et al. (1998) fed seabass (initial wt 14 g) 

with diets containing fish meal (68.6% of dietary protein) or soybean meal (65.6% of dietary protein). 

Supplementing the soybean meal diet with 1000 and 2000 units of microbial phytase per kg diet increased apparent 

P absorption 72% and 80%, respectively. Vielma et al. (1998) fed rainbow trout (initial wt 52 g) in excess with 

diets containing 0 or 1500 units phytase per kg diet, and 2500, 250,000 or 2,500,000 IU cholecalci ferol per kg diet 

for 12 weeks. The basal diet provided 5.8 total P and 3.2 g phytate P/kg dry matter. Soy protein concentrate was 

the primary source of the phytate P. Weight gain of fish was increased by phytase but decreased by high dietary 

cholecalci ferol. Phytase increased apparent availability of P and bone ash, plasma and body P concentrations. 

Dietary cholecalci ferol levels did not influence P utilization. Schroder et al. (1996) indicated that, in pigs, about 

70% is the upper limit of digestibility of P in plant materials supplemented with microbial phytase. General 

reviews on phytase in human and animal nutrition have been reported previously (Cosgrove 1980, Nayini & 

Markakis 1986, Jongbloed et al. 2000). 

Optimum Ca/P ratio 

Lipschutz (1910) found that dietary Ca to P ratio is important in dogs. A ration very low in P content produced a 

moderate increase in weight of the dog, but after 7 weeks, there were protracted muscular clamps and other 

physiological derangement. A ration containing the same amount of Ca but more P caused normal development. 

Sherman & Pappenheimer (1921) found that the addition of Ca-lact ate (3%) to a low-P diet produced rickets in 

rats. McCollum (1923) emphasized that the ratio between Ca and P in a diet is very important in producing rickets 

in rats. Diets low in Ca and high in P developed rickets that was complicated with tetany. Diets low in P and high 

in Ca also developed rickets but not tetany. The composition of a diet that produced the most extreme degree of 

rickets in young rats were as follows; wheat (whole) 33%, maize (whole) 33%, gelatin 15%, wheat gluten 15%, 


34

NaCl 1%, and CaCO 3 3%. The Ca/P ratio is also important in analysis. Lehmann (1851) wrote, "The 

phosphorus exists chiefly in the yolk, where it occurs as glycero-phosphoric acid, which during incubation is 

gradually decomposed, so that the liberated phosphoric acid unites with lime which passes over by endosmosis from 

the shell into the egg to form this salt. There is, however, so much phosphorus contained in the yolk of the egg, 

that on incineration it forms acid phosphates, or rather metaphosphates, with the bases which it there encounters (p. 

418)." This seems to be unknown to some contemporary researchers in biology since total P content of some 

analyses, especially of high-P-low-Ca (or Mg) ingredients or diets is sometimes reported to be too low. They are 

not to be incinerated at ordinary analytical temperatures (500-600ºC). 

Phillips et al. (1958) and Phillips (1959) concluded that the optimum Ca:P ratio for maximum P retention (in 

24 h) was 1:1; however, in a later study Phillips et al. (1959) obtained the best retention of P (in 4 d) with synthetic 

diet containing no Ca supplement (Ca:P ratio of 0:1). Sakamoto & Yone (1973) reported that juvenile red 

seabream grew better when casein-gelatin diet (0.34% Ca level) contained 0.68% total P (forti fied with monosodium 

phosphate) than when the diet contained lower (0.19, 0.34%) or higher (1.36%) total P. The optimum Ca/P ratio 

was 1/2. Reducing dietary Ca level but keeping the same P level (Ca/P ratio 1/5) markedly lowered growth of the 

fish. High dietary P levels or low Ca/P ratios reduced appetite of the fish. Dietary P levels or Ca/P ratios little 

affected blood total P levels (range 1150-1350 ppm); however, they increased Pi levels in the blood serum from 125 

(0.19%P/diet) to ca. 200 ppm (0.68 and 1.36%P/diet). Nose & Arai (1979) also reported that Japanese eel requires 

both Ca and P in the diet for optimum growth. Lovell (1978) did not see any significant effect of Ca/P ratio on the 

weight gain of channel cat fish. IOM (1997) states that the Ca:P ratio by itself is of severely limited value, in that 

there is little merit to having the ratio "correct" if the absolute quantities of both nutrients are insufficient to support 

normal growth. 

Precipitation of available P by Ca and other cations 

The absorption of inorganic P is intimately related to the amount of Ca in the diet, or other cations which form 

insoluble salts in the intestinal tract, and thus related to the absorption of Ca as well (McCollum et al. 1939, 

Hegsted 1960). Bondi (1987) also wrote, "Excess Ca in the diet reduces the absorption and utilization of minerals, 

particularly of P and trace minerals." In 1878, Bertram found that CaCO 3 in diets served to deflect P from urine to 

feces. Herxheimer (1897) also noted that when CaCO 3 was baked into bread at 5%, urinary P decreased quite 

markedly, while feces P increased. Paton et al. (1899-1900) wrote, "Lime salts have been supposed to form 

insoluble compounds with phosphoric acid in the intestine and thus to prevent its absorption." Bergmann (1901) 

also noted that while dogs normally excrete P mostly in the urine, if Ca is abundant in the food much P is excreted 

with it in the feces. Keller (1899, 1900) showed that the addition of fat in the diet increases the excretion of P in 

the urine, and decreasing it in the stool. He thought that this partition is brought about by the formation of fatty 

acids which combine with calcium to form soaps. The calcium is excreted in the stool as phosphate and as calcium 

soaps, mainly as calcium oleate, for the formation of which about 1 part of CaO is required to 10 of the fatty acid. 

This absorption of the calcium by the fatty acids sets free the phosphoric acid and allows it to be absorbed by the 

intestine, as is evidenced by its increase in the urine. Forbes & Keith (1914, pp. 212) based on available 

evidence by that time concluded "The ingestion of alkaline earths decrease urinary P elimination by uniting with P, 

both food and metabolic, in the intestine to form diffi culty soluble salts, thus hindering P absorption. With a Ca-poor 

diet the feces are poor and the urine is rich in P." Other cations such as iron (Waltner, 1927) and aluminum 

(Deobald & Elvehjem, 1935) also form insoluble compounds with P. Bishop & Williams (1958) reported feeding 

chicks with aluminum hydroxide gel caused a syndrome of weakness leading to death within a week after onset. 

This was associated with low plasma Pi and blood ATP, which was prevented by the injection of sodium 

monophosphate. In humans, Lotz et al. (1968) reported that intake of aluminum-cont aining antacids causes a loss 

of P from the body. NRC (1983) states that aluminum toxicity is primarily due to its property to reduce intestinal 

P absorption. Phillips et al. (1958) fed brook trout with synthetic diets containing 32 P with varied levels of Ca (i.e., 

varied Ca/P ratio). When the diet was free of Ca, fish completely absorbed dietary P within 24 h, and excreted 

64% of the absorbed P into water (probably via urine). When Ca/P ratio was 1/1, about 15% of dietary P remained 

unabsorbed 24 h aft er feeding, but fish retained somewhat more P than when the diet was free of Ca. When Ca/P 

ratio was further increased to 4/1, 40% of dietary P remained unabsorbed after 24 h, and the fish retained the least 

amount of P. Phillips et al. (1959) in a 4 day trial noted that dietary Ca progressively reduced dietary P utilization. 

The Ca/P ratio of 0/1 provided the best utilization (retention) of dietary P. Phillips et al. (1960) confirmed this 

observation. Phillips et al. (1964) showed that the retention of dietary 32 P (as H 3PO 4) was higher when a synthetic 

diet was free of Ca than when the diet contained Ca at a level of 1/1 in Ca/P ratio in both low P diet and high P diet. 

Dietary Ca also caused the retardation of P absorption from the diet. Muscle Pi was gradually incorporated into 


35

organic forms through 4 days after feeding, however, at the 4th day, predominant portions (ca. 92%) of 32 P in muscle 

were still in the inorganic fraction rather than phospholipid, nucleic acid, or protein fractions. The muscle 

contained 1.40 mg Pi, 0.18 mg phospholipid-P, 0.25 mg nucleic acid-P, and 0.10 mg phosphoprotein-P per gram. 

Hurwitz et al. (1978) determined the effect of dietary Ca levels on the apparent absorption of P by measuring P and 

radioactive yttrium content in the feed and feces of turkey. Dietary Ca intake over 440mg/d reduced the fractional 

absorption of supplementary P in a linear manner. Nakamura (1982) fed carp for one week with 

egg-albumin-bas ed diets of varied Ca content (0.1, 0.4, 0.7, 1.3 and 2.6%) but fixed P content (0.64%). Ca-lactate 

and KH 2PO 4 served the sources of Ca and P in the diets, respectively. Feces were collected by stripping. The 

highest absorption of P (98.1%) was observed with a diet of the lowest Ca content (0.1%). The P absorption 

decreased as the dietary Ca level increased. Vielma & Lall (1998b) fed Atlantic salmon (initial wt 42 g) for 15 

weeks with a low-P diet (3.1 g P, 1.3 g Ca /kg diet) with or without Ca (as CaCO 3) and/or P supplementation. The 

P in the basal (low-P) diet was supplied solely from casein, while inorganic P supplied a large portion of P in the 

high-P diet. Thus, the sources of P in the low-P diet and high-P diet were different, and so does their interaction 

with Ca. The vertebral P content of fish after 15 wk of feeding did not differ between fish fed the low-P (basal) 

diet (3.1 g P/kg diet) and fish fed high-P diets (8.3 g P/kg diet) when the diets were supplemented with Ca. This 

indicates that the dietary P requirement was about satisfied at 3.1 g P/kg diet based on bone P content. The 

authors found that supplementing the low-P diet with Ca greatly increased bone calci fication, but decreased weight 

gain. Retention and intestinal absorption (digestibility) were not determined. They wrote, ". . . from the practical 

diet formulation point of view, too high of a dietary Ca content per se seems not to be of a concern (for dietary P 

utilization)." This conclusion, however, seems to disagree with the other studies discussed above. Walter et al. 

(2000) fed rats on corn-soybean diet. Increasing the supplementary level of calcium carbonate proportionately 

reduced apparent P absorption by the rat. The apparent Zn absorption and femur zinc concentration were also 

decreased with calcium supplementation to the diet. The reduced P absorption might be due to the formation of 

phytate-Ca that precipitates in the intestinal lumen and is neither absorbed not digested by luminal phytases. 

Adverse effects of excess P 

Numerous studies in animals and fishes showed that the absorption or bioavailability of dietary trace minerals (e.g., 

Zn, Mn) is decreased by excess Pi in the diet. Milby (1933) and Hammond (1936) noticed and Wilgus et al. 

(1937) and Wiese et al. (1938) confirmed that dietary excess calcium phosphate induces perosis in the chick. 

Wedekind et al. (1991) showed that excess P per se is the antagonizing factor of Mn absorption. Baker & Oduho 

(1994) repoted that excess dietary P is anorectic unless Ca is added to the diet to keep the ratio adequate. Hossain 

& Furuichi (2000a; 2000bc) reported that scorpion fish and Japanese flounder required calcium in diet for optimum 

growth. The basal diet contained about 1.0% available P supplied from casein and monosodium phosphate, which 

is considerably high compared with the dietary requirement of P (about 0.6% for most fishes). Interestingly, the 

fish fed diets low in Ca, though growth was suboptimal, had similar body and bone P and Ca contents. Hossain & 

Furuichi (1998) reported similar results with puffer fish. If a high level of available P in diet has any adverse 

physiological effect, an increas ed growth rate by calcium supplementation should be attributed to as due to reduced 

intestinal P absorption rather than to the nutritional essentiality of calcium. Since the capacity of renal P handling 

is limited, high intakes of dietary P could lead to hyperphosphatemia, unless P is precipitated by Ca in the intestinal 

lumen. Dietary Ca might effectively reduce the toxicity of excess dietary (availabl e) P to the organism. Dietary 

calcium or antacid is a means to reduce renal burden. Also, the seawater is abundant in Ca that fish can absorb. 

Thus, the Ca supplementation might be unnecessary if the basal diet is normal in P content. In rainbow trout, 

Sugiura et al. (2000) showed that when dietary available P was higher than the requirement level in a semi-purified 

low-calcium diet, the N retention-- i.e., growth rate-- of the fish was depressed. This subject has been discussed in 

Ketola (1979), Gatlin & Wilson (1984), Richardson et al. (1985), Hardy & Shearer (1985), Satoh et al. (1987, 

1989, 1992, 1996, etc.) for fish, and in Ammerman et al. (1995), Mertz (1987), and other textbooks for animal 

species. 

Physiology of P transport 

Dietary P absorption occurs in the upper part of the small intestine in humans, many animals and birds (Cross et al. 

1990, Breves & Schroder 1991, Danisi & Murer 1991, Schroder et al. 1996), whereas the P transporter protein 

seems to be most abundant in the ileum (discuss later). This discrepancy is due to paracellular diffusion that is 

dominant in the duodenum. Murdoch (1927) and Warkany (1928) report ed that infants were able to bring about 


36

an exceptionally rapid absorption (peaked within one hour) of P from the alimentary tract following the oral intake 

of NaH 2PO 4 or Na 2HPO 4. Anderson (1991), Anderson & Barrett (1994), and Groff et al. (1995) also mentioned 

that orally administered 32 P in humans appears in the blood within 10 min and peaked after about an hour. Phillips 

et al. (1961) suggested that dietary P absorption occured in the stomach as well as in the intestine based on the 

observation that radio-labelled P was rapidly recovered in the blood of brook trout (within a few hours). Podoliak 

& McCormick (1967) fractionated acid-soluble P in muscle and viscera into ionic P, which is Pi, and non-ionic P, 

that included labile organic P such as ADP, ATP, PCr, and many others. Most of the labeled P recovered in muscle 

and viscera at 12 hours and through 4 days after feeding was non-ionic, i.e., some labile organic P. They concluded 

that P coupled with a carrier (possibly ADP) in either stomach or the intestine is the major path of P absorption and 

transportation of dietary P in brook trout, while some ionic P (Pi) could be involved in the initial absorption through 

the stomach wall. Podoliak & Smigielski (1971) repeated the experiment using rainbow trout and brown trout. 

Dietary 32 P was rapidly absorbed from the intestine with concomitant synthesis of acid-soluble non-ionic P in the 

tissues of the GI tract. In muscle, however, the amount of 32 P recovered was much lower, and the ionic form was 

dominated. The labile (acid-soluble non-ionic) P in the GI tract was supposed to mediate the effici ent P transfer to 

the tissues. 

Hurwitz et al. (1978) found in turkeys that P absorption is neither saturable nor regulated at concentrations 

encountered in the intestine in vivo. They wrote, "The linearity of phosphate absorption as a function of intake, 

suggests no adaptation of the phosphate transport system to either low or high intakes of phosphate. Thus, in 

contrast to calcium and sodium, the absorption of phosphate appears not to respond to the physiological needs." 

Also, according to IOM (1997), "there is no apparent adaptive mechanism that improves phosphorus absorption at 

low intakes . . . A portion of phosphorus absorption is by way of a satuable, active transport facilitated by 1, 

25-dihydroxyvitamin D (1,25(OH) 2D). However, the fact that fractional phosphorus absorption is virtually 

constant across a broad range of intakes suggests that the bulk of phosphorus absorption occurs by passive, 

concentration-dependent processes. Also, even in the face of dangerous hyperphosphatemia, phosphorus continues 

to be absorbed from the diet at an effi ciency only slightly lower than normal." (see chapter "Errors due to high 

dietary P levels") Nakamura (1985), using stripped and everted intestine of the carp, demonstrated in an in vitro 

experiment that Pi absorption occurs in all parts of the intestine, with the highest rate in the middle section. This 

result agreed with an in vivo study in which Pi concentrations in the luminal fluid of the intestine of the carp fed a 

diet containing Pi at a level of 0.64% decreased along the intestine. Avila et al. (2000) studied intestinal 

P-transport mechanisms of rainbow trout. They found that the intestinal P-transport is the sum of Na-dependent 

saturable process and Na-independent passive diffusive process. At low luminal Pi concentrations, the former 

process predominates, whereas at high luminal Pi levels the latter process becomes more important in intestinal 

Pi-uptake in trout. The active Na-dependent process also were shown to be depressed in trout acclimated for 28 

days with diets containing high levels of P. The Pi-uptake was almost two times higher in the proximal and 

middle-parts than the distal part of the intestine. Mol et al. (1999) reported that high Ca and/or Mg content in 

water can reduce P-uptake from the intestine, and thereby reduce body P level in catfish. 

In fish, the physiological mechanisms of dietary P absorption and body P regulation have not been well 

studied. In monogastric mammals, dietary P is known to be absorbed via two channels; i.e., (1) active 

career-mediated transport via NaPi and (2) passive paracellular di ffusion that is not saturable regardless of the 

luminal P concentration. The active transport is a sodium-dependent transcellular process (Pi is transported from 

the intestinal lumen across the apical brushborder membrane of the enterocytes; from the cell, exit across the 

basolateral membrane into the blood). The low K m relative to the luminal P concentration greatly limits the 

contribution of this pathway in total Pi transport. The passive transport is non-regulated, and even at high dietary P 

intakes, P is continuously absorbed at relatively high rate. However, paracellular perm eability in the intestine 

increas es via a cAMP dependent pathway as well as other pathways that involve PKC and other factors. For 

example, calcitriol increas es paracellular Ca diffusion as well as transcullular Ca uptake in mammals. 

Physiological regulation of paracellular permeability remains to be studied. In the kidney, Pi is filtered through the 

glomerulus and NaPi-IIa cotransporter, isoform of NaPi-IIb of the intestine, located on the apical surface of the 

proximal convoluted tubule reabsorbs Pi from the filtrate back into the renal cells, and then from the cells into the 

blood across the basolateral membrane. P that has not been reabsorbed will be excreted in urine. In fish, however, 

Pi is also secreted into the tubular lumen via a Na-dependent mechanism. 

Three families of NaPi cotransporters are identi fied and named type-I, type-II and type-III NaPi 

cotransporters. The three families of NaPi share no significant homology in their amino acid sequence. Their Pi 

affinity, pH dependence and tissue expression are unique. Type-II NaPi is known to be the most important P 

transporter in the intestine and kidney (see reviews Takeda et al. 1999, Murer et al. 2001). In mammals, IIa is 

renal, IIb is intestinal, and IIc is renal age-relat ed isoforms of the type-II NaPi. In fish, both renal and intestinal 


37

isoforms are IIb (see Werner & Kinne 2001), but the renal and intestinal isoforms are fairly different in sequence, 

except flounder that has an identical NaPi in both tissues. Pi uptake stimulator (PiUS) or inositol hexakisphosphate 

kinase (Schell et al. 1999) may be involved in the absorption of dietary P. Norbis et al. (1997) first isolated PiUS 

mRNA from rabbit duodenum. Katai et al. (1999) subsequently isolated the corresponding sequence from rat 

small intestine. These PiUS cRNA markedly increased Na-dependent Pi-uptake when injected into Xenopus laevis 

oocytes. Katai et al. (1999) further described that dietary P restriction (7 days) up-regulated PiUS genes (~2 times, 

per mRNA abundance) and Pi uptake from BBMV (~2 times) in the rat intestine. In fish, PiUS in both the intestine 

and kidney appears to be only slightly responsive to chronic dietary P restriction. 

Dietary regulation of Pi transport and NaPi-II mRNA/protein expression in the intestine have been studied 

in mammals (Hattenhauer et al., 1999; Katai et al., 1999; Huber et al., 2000; Huber et al., 2002). In mammals, 

the main site of dietary P absorption is the proximal small intestine (Danisi & Murer 1991), and unlike kidney, 

apical expression of NaPi-IIb prot ein in the small intestine, is mostly in response to longer term (days) situations 

(Murer et al. 2001). In rainbow trout, Avila et al. (2000) reported that active Pi transport is higher in the proximal 

than distal intestine. They also have functionally characterized P-transport in trout using everted intact intestinal 

sleeves. It was shown to have a saturable, career-mediat ed active component and a diffusive non-saturabl e 

component. The study also showed that the dietary Pi level did not affect Pi-uptake rat e in trout intestine at day-7, 

but significantly down-regulat ed Pi-uptake at day 28, indicating that the response of NaPi-II in trout intestine to 

dietary P concentration is not acute, but chronic. They did not measure NaPi-II protein or mRNA abundance of the 

tissues. Avila et al. (1999) incubated everted trout intestine for one hour in a Ringer solution containing either 

vitamin D3, 25(OH)D or 1,25 (OH) 2D; however, none of these pre-incubation did increase 32 P uptake from the 

intestine. 

Pyloric caeca (PC) are finger-like diverticula stemming from the duodenal region of the small intestine of 

many fish species. The physiological functions of PC have not been well researched; however, they are thought to 

be the auxiliary to the proximal small intestine because of their histological simalities. Buddington & Diamond 

(1987) studied PC of rainbow trout: It has about 56 PC, which collectively contribute about 70% of the total gut 

surface area. They also studied the contribution of PC to the total uptake capacity (of the entire gut) for glucose, 

the dipeptide carnosine, and nine amino acids, which ranged between 68 and 81%. Thus, the functional 

contribution of PC corresponds well to its contribution to the total gut surface area. A recent study has shown that 

Pi absorption in trout PC is largely a passive paracellular process facilitated by high luminal Pi concentrations. 

Approximately 89% of total Pi absorption takes place in PC, and about 92% of the Pi absorption in PC is diffusive at 

physiological luminal [Pi] of 20 mM (i.e., luminal [Pi] when a normal P diet is fed). This explains the fractional Pi 

absorption, which is continuous over the wide range of dietary P levels, even at concentrations much higher than the 

dietary requirement. The active Pi absorption is modulated by several factors, including temperature, luminal Pi 

concentration, fish P status, and in particular luminal pH. An isoform of NaPi isolated from trout PC is similar to 

the intestinal NaPi in base sequence, but functionally different one another. 

NaPi transporters are sodium-dependent symporters. Zebrafish and flounder NaPi-IIb are 

Na+-dependent. Trout intestinal NaPi-IIb is Na+ dependent (Avila et al., 2000). Trout PC NaPi is 

Na+-dependent at alkaline pH, but not at neutral pH. Pi uptake in PC is much higher at alkaline pH, and this 

increas e is strictly Na+-dependent, which is similar to that of the mammalian renal NaPi isoforms (Hilfiker et al., 

1998; Murer et al., 2001). Danisi & Murer (1991) reported Na+-independent, active (carrier-mediated) Pi 

transport in rat and chick intestinal basolateral membrane and in dog renal basolateral membrane; however, the 

molecular identity of the transporter is unknown. Mammalian intestinal NaPi isoforms have higher transport rates 

at neutral to acidic pH (Berner et al., 1976; Borowitz & Ghishan, 1989; Danisi et al., 1984; Lee et al., 1986; 

Tenenhouse, 1999; Xu et al., 2002). Thus, intestinal NaPi are functionally different between mammals and fish. 

However, in chick, pig and sheep intestines, Pi transport rate is higher at alkaline pH than acidic pH (Danisi & 

Murer, 1991), which is similar to intestinal and renal NaPi isoforms of zebrafish, flounder and trout (Forster et al., 

1997; Graham et al., 2003; Kohl et al., 1996; Nalbant et al., 1999). Since at pH higher than 7.2 divalent ions 

2– – 

(HPO4 ) will be the dominant species than monovalent ions (H2PO4 ), the preferred ions for the fish NaPi isoforms 

and mammalian renal NaPi isoforms may be the divalent ion, whereas for mammalian intestinal isoforms, the 

preferred species may be the monovalent form. 

Dietary Acidification and P availability 

Heitzmann (1873) stated that lactic acid increased in the course of rickets and brought about dissolution of the salts 

of the bones. Anderson (1878) hypothesized about the etiology of scurvy as follows; "The conditions 

characteristic of this disease (i.e., scurvy) are always accompanied with a deficiency in the food of the materials for 


38

the form ation of tissue phosphate; or if these materials be present in suffici ent quantity, examination of the food in 

use shows that the salts, which enter into the formation of tissue phosphate, are in an insoluble, or only partially 

soluble state (p. 108)." Also, he said, "The action of citric acid is remarkable. It is a complete and perfect solvent 

of phosphates; even tricalcic phosphate (bone phosphate) is in certain proportions completely dissolved by it (p. 

115)." and said further "Citric acid also prevents the formation of insoluble phosphates of iron, magnesia and 

alumina . . . and the consequent loss of phosphoric acid required for the formation of tissue phosphate. It 

economises phosphoric acid and bases for the fabri cation of tissue phosphate for purposes of nutrition, by preventing 

the loss of phosphoric acid, which might otherwise form insoluble phosphates, useless in nutrition . . . the efficacy of 

lime juice as an anti-scorbutic depends mainly on the presence of citric acid, and is referable to the solvent powers 

of this acid on phosphates. (p. 117)." 

Baginsky (1882), a pediatrist in Berlin, fed puppies two grams of lactic acid daily, and reported that the total 

ash of the bones was decreas ed to an extent greater than when the acid was not fed. Weiske et al. (1885) fed hey 

acidified with sulfuric acid to sheep for 4 months, but did not see any effects on bone composition. In a later work, 

Weiske (1891) report ed that sulfuric acid, if ingested for a considerable time, lowered Ca content of both bones and 

muscles. Shohl & Sato (1923) reported that when an infant was given 250 cc of 0.1N HCl the amount of calcium 

increas ed in both the urine and feces. Wills et al. (1926) also reported that when hydrochloric acid milk, 

containing 20% of 0.1N acid, was fed, the calcium retention was decreased and the balance became negative. 

Ammonium chloride also failed to improve the balance. Hess (1929) wrote that one of the most important 

mechanisms for regulating the acid-bas e equilibrium of the body is the excretion of phosphate by the kidneys. 

Sherman et al. (1936) found that the dog oxidizes an average 99.3% of citric acid administered orally when 0.5-2.0 

g of citric acid were given per kg of body weight. The unoxidized citric acid (0.7% of intake) appeared in the urine, 

and none found in the feces. Wright & Hughes (1976) reported dietary levels of citric acid at 4-5% (ca.3 g/kg/d) 

had no observable toxic effects in guinea pigs and rats compared with the control animals fed non-acidi fied diets in a 

60 day feeding trial. Møllgaard (1946) showed that oxy-acids, such as tartaric, citric and lactic acids, shift the 

precipitation point of calcium phytate (pH 3-4) to a more alkaline reaction, which considerably improves the 

absorption of Ca and P from the intestine of pigs. Organic ligands, such as citric acid and EDTA, chelate minerals 

(e.g., Ca, Fe, Zn), which otherwise precipitate phytate P and Pi in the intestine (Vohra & Kratzer 1964; Nielsen et al. 

1966; Lyon 1984). Phytate-mineral complexes, which are insoluble at pH higher than 7, have higher solubilities at 

lower pH (Tangkongchitr et al. 1982; Grynspan & Cheryan 1983; Nolan et al. 1987). Bonting (1952) studied 

long-term effects of citric acid and phosphoric acid intakes on the regulation of acid-bas e equilibrium using rats. 

The basal diet contained casein, dried milk, wheat flour and potato flour as the major ingredients, and the sources of 

P were tricalcium phosphate and disodium phosphate. Citric acid (1.2%) supplementation did not change fecal and 

urinary excretions of P and the balance. Phosphoric acid (0.4%) supplementation increased the P balance and the 

urinary P excretion markedly, while the fecal P was not increased. Calcium excretions, both fecal and urinary, and 

the balance of Ca and N did not change appreciably by both acids. Day (1940) reviewed citrates and its 

anticalci fying action. Hegsted (1960) briefly discussed about dietary citric acid intake, intestinal acidity, and 

among other factors that might affect Ca absorption in humans. Kirchgessner & Roth (1980) reported that 

apparent absorption of Ca, P, and Zn were improved by 13, 11, and 33%, respectively, and the balance of these 

minerals in the order named was also improved by 14, 13, and 43% by the addition of 2% fumarate (sic) in weaning 

pigs. The author suggested the chelating property of fumaric acid with various cations increases the absorption of 

the metal-fumarate complex. Radecki et al. (1988) found that the addition of fum aric acid (1.5%) to a 

corn-soybean meal diet improved gain and feed efficiency of weaning pigs; however, citric acid did not produce 

such effects. The authors also found that fumaric acid increased fecal Ca, P and Zn, and reduced urinary P. The 

retention or balance of Ca and P did not change; however, the retention of Zn decreas ed. Effects of dietary 

acidification on the general performance of piglets have been studied extensively (reviewed in Ravindran & 

Kornegay 1993). It has been shown that dietary citric, lactic, acetic, propionic and other organic acids have an 

anorectic (hypophagic) effect and reduce fat deposition in broilers when fed 3-5%, both of which seem to be 

desirable effects (e.g. Lessard et al. 1993). 

Many researchers of livestock animals and poultry confirmed significant effects of dietary acidi fication on 

animal growth and feed effici ency; however, the mode of action is still uncertain. It should be noted that the basal 

diets used in those animal studies were formulated with plant ingredients and without fish meal or animal by-product 

meals. Fish feeds generally contain substantial amounts of fish meal, which supplies bone phosphates that are not 

very effici ently absorbed by the fish, especially agastric fishes such as carps. Thus, the mode of effect of dietary 

acidification appears to be different between animal feeds and fish feeds. Rungruangsak & Utne (1981) prepared 

fish mince-based moist diets acidified with hydrochloric acid, formic acid, or sulfuric acid (2.5%, w/w). All diets 

were further supplemented with 0.5% propionic acid as a mold inhibitor. The dietary pH in the order named was 


39

4.4, 4.1 and 3.3. The acidified diet was fed to rainbow trout (95-130 g in body wt) for 140 days either solely or in 

combination with the control diet (non-acidified, pH 6.6) at various ratios. These authors found that 1.5-2.5% 

dietary acid levels depress ed feed intake and growth of the fish. At 1.25%-level of acidi fication, the dietary pH 

was 5.3 (hydrochloric acid), 4.6 (formic acid), and 4.3 (sulfuric acid). The feed intake of fish did not differ, but 

feed conversion was low in formic acid and sulfuric acid groups. Fish growth was also low in formic acid and 

sulfuri c acid groups in the first month compared with non-acidified and hydrochlori c acid groups; however, all 

groups showed similar growth during the second and third months. Asgard & Austreng (1981) reported that 

formic acid alone or in combination with sulfuric acid is, from practical and economical reasons, best suited to make 

acid silage of fish offal. Hardy et al. (1983) studied the effects of acidi fication. Acidified fish meal and acidi fied 

fish silage both slightly reduced growth and feed effi ciency of rainbow trout in 84 days of feeding compared with 

those neutralized with Ca(OH) 2. Those wet materials were acidifi ed with 2% sulfuric acid and 0.75% propionic 

acid, which after drying and mixing with other materials concentrated to 5.9% of the diet. Thus, the diet acidity 

seems to be very high compared with other acidification studies especially because strong acids like sulfuric acid are 

almost completely dissociated. This study indicated that the fish fed the acidi fied (non-neutralized) diets had 

higher P and Ca contents in the body than the other fish fed neutralized diets (though the authors did not mention 

this), suggesting acidification increased availability of dietary P. This also suggests that the silage may be used 

without neutralization, and the diet may be prepared as moist pellets and stored at ambient temperature. 

The advantage of dietary acidi fication is that the acid can increase P digestibility, which was not a matter of 

interest in the past. Vielma & Lall (1997) reported that rainbow trout increased absorption of P, Ca and Mg when 

a fish meal-based diet was supplemented with formic acid (0, 4, 10 mL/kg). However, the level of increase was 

small (70% P-absorption in non-acidifi ed diet vs 75% in acidified diet). The dietary pH was 6.3 and 5.3 in 

non-acidi fied and acidified diets, respectively. However, the pH of the intestinal contents (both proximal and 

distal) of the fish fed the acidi fi ed diet was higher than that of fish fed the nonacidi fied diet. Vielma et al. (1999) 

fed rainbow trout (initial wt 3 g) for 6 weeks with casein-based diets containing varied levels of bone meal with or 

without supplemental citric acid. The diet contained total P in the amount of 0.5-0.6%; and citric acid was added in 

the amounts of 0, 0.4, 0.8 and 1.6%; which resulted in the dietary pH of 6.0, 5.7, 5.4 and 5.0, respectively. Dietary 

supplementation of citric acid increased whole-body concent rations of ash, P, and Ca, but only modestly (0.297 vs 

0.313%P in non-acidified vs the most-acidified diets, respectively). Sugiura et al. (1998) reported that 

supplementing a fish meal-based diet with citric acid at a level of 5% increas ed P absorption from 75% (non-acid 

diet) to 87% in rainbow trout. However, sodium citrate had no effect, and sodium bicarbonate decreased the 

apparent P absorption. Fecal P content of fish fed acidifi ed diets was about 1/3 of the non-acidified group. 

Supplementing a high-ash diet (2.0%P) with citric acid increased urinary P and decreased fecal P, with no change in 

body P retention in fish. Thus, fecal P can be reduced by increasing P availability (by acidification), while urinary 

P must be reduced by reducing P content in the diet. Inorganic acids also increased availability of P in fish 

meal-bas ed diet from 71% (non-acidi fied, pH 6.1) to 95% (sulfuric acid, pH 2.4) or 88% (hydrochloric acid, pH2.0). 

The fish accept ed the acidic diets during 23 days of satiation feeding with slight (sulfuric acid) or some 

(hydrochloric acid) reduction of feed intake. Adding citric acid to a soybean meal-based diet containing no fish 

meal had without effect on P availability. However, P availability increased markedly when both citric acid and 

phytase were added compared with when only phytase was added to the diet. Radcliffe et al. (1998), however, did 

not see any synergistic interaction between dietary phytase and citric acid in corn-soybean meal-bas ed diets fed to 

weaning pigs. Also, the addition of citric acid alone had little effects on performance and Ca digestibility. Some 

workers observed a reduction of intestinal pH in pigs fed acidi fied diets (Scipioni et al. 1978, Burnell et al. 1988), 

which may prevent minerals from being precipitated and phytase from being inactivated in the intestinal lumen. 

Thus, low intestinal pH may confer favorable luminal environment for P absorption. However, intestinal 

sodium-phosphate cotranspoters (type-II) have a strong pH preference. In mammals, they are generally more 

active at acidic pH, whereas in fish they are much more active at alikaline pH (see “P transport” section). The 

intestinal pH of fishes is strongly alkali (especially of seawater fishes) compared with homeotherms due presumably 

to low bacterial fermentation in the intestinal lumen of fishes (Wilson et al. 2002). In fish intestine, therefore, the 

NaPi transporter is very active, whereas at the same time P tends to precipitate. However, high bicarbonate 

concentrations in the lumen of fish intestine may protect some phosphatre salts from being precipitated. Maier & 

Tullis (1984) noted that lowering dietary pH (by inclusion of acetic acid) or raising the pH (by ammonium 

hydroxide) of an algae-based diet had a significant effect on the gut pH of tilapia. Chonan et al. (1998) reported 

that the absorption of dietary Ca and P increased in gastrectimized rats when lactic acid was added to the diet. 

Jongbloed et al. (2000) studied the effect of lactic acid (1.6 or 3.2% /diet) and formic acid (0.8 or 1.6%) 

supplementation to a pig diet with or without microbial phytase. Both phytase and acids increased P digestibility; 

however, there was no interaction. The acid level did not affect the P-digestibility. The acidification did not 


40

increas e urinary P. Boling et al. (2000) reported that both citric acid and sodium citrate (6%/diet) markedly 

increas ed bone density, weight gain and feed intake (but not feed efficiency) of chicks fed P-defi cient corn-soybean 

diet. The effect was absent with phytate-free (casein-dextrose) diet. Conversely, pigs fed P-deficient 

corn-soybean diet increas ed neither bone density nor weight gain by dietary addition of citric acid; but greatly 

improved feed effi ciency (gain/feed). Many researchers have postulated modes of action of citric acid or citrates 

on phytate-P utilization. Based on available evidence, they argue that dietary citrates have an effect to chelate Ca 

in the intestinal lumen and prevent precipitation of phytate-P (discussed in Boling et al. 2000a; see sections 

“Mellanby’s toxamin” and “ Availability of phytate-P”). Ca-phytate is resistant to phytase hydrolysis, while soluble 

phytates (phytic acid and sodium salts) are digestible by phytase (endogenous, bacterial and ingredient-derived). 

Therefore, as Boling et al. (2000b) demonstrated, when a diet is high in Ca content, in the amount that overwhelms 

the chelating capacity of citrates, then dietary addition of citrates has no effect on phytate-P utilization. 

A recent study indicates that dietary acidification (with inorganic acid) inhibits gastric H+/K+ ATPase 

(proton pump) and sodium bicarbonate cotransporter gene expressions in corpus stomach of rainbow trout. This 

suggests that gastric acid secretion may be down-regulated by dietary exogenous acid intake by some feed-back loop 

mechanism, which involves not only decreased proton secretion into the gastric lumen, but also corresponding 

decrease of basolat eral bicarbonate exit (from the oxynticopeptic cells), leading to a deficit of bicarbonate needed 

for gastric mucous cells as well as for neutralizing gastric chyme in the duodenum or pyloric ceaca. Thus, the 

inhibition of proton pump by dietary inorganic acids could lead to an acid-base imbalance. Dietary acetic acid, 

however, seems to be neutral in this regard. 

In vitro estimation of P availability 

Some earlier workers in the mining field apparently used simple laboratory techniques that could estimate P 

"assimilability" of rock phosphates that was to be used as P supplements for animal feed (Raynolds et al. 1944). 

The supply of bone meal, which was a common P supplement for animal feeds, was rather limited in those days. 

The methods they used to evaluate the biological value of P in rock phosphates were based on the solubility of P in 

dilute hydrochloric acid within the range of concentrations found in the stomach of animals. Reynolds et al. 

(1944) and Hill et al. (1945) appears to be the first to conduct extensive investigations to estimate P availability in 

various P supplements based on their solubility in dilute hydrochloric acid and other solvents. Gillis et al. (1948) 

studied 19 P compounds in an in vivo chick bioassay (at 0.4% and 0.8% levels), and an in vitro 0.4%-HCl solubility 

test. Correlations between in vitro and in vivo assays were low, which was probably because the study included 

varieties of metaphosphates, pyrophosphates, phytate-phosphate and inorganic orthophosphates. Also, Day et al. 

(1973) compared bioavailability of P compounds for chicks by an in vivo bone-ash assay and by in vitro solubility 

tests using 0.4% HCl, 2% citric acid and neutral ammonium citrate. Satoh et al. (1986) and Shinma (1989) 

reported in vitro methods for estimating phosphorus availability in various feed ingredi ents and commercial feeds 

for fish based on differential solubility fractionation using distilled water, 80% acetic acid, and 0.9% hydrochloric 

acid as the solvents. They noted that carp could utilize water-soluble fraction of dietary P, but trout could utilize all 

three fractions of dietary P. Satoh et al. (1992) and Satoh et al. (1997) reported an in vitro technique to estimate 

available P content in semi-purified diets, practical diets and commercial diets for carp and rainbow trout. The 

values agreed well with those determined in in vivo feeding (digestibility) trials. Deionized water, 80% actic acid 

and 0.25M-HCl were used to extract phosphorus from the testing materials. The extracted solutions were digested 

with nitric-perchloric acid mixture. The authors did not discuss about the interference of phytate-P, which is highly 

soluble in water and even more soluble in dilute acid (Jordan et al. 1906, Han 1988), but not available to fish. 

Jahan et al. (2000) from the same laboratory also reported that the in vitro (solubility test) and in vivo (digestibility 

trials) data of estimated P availability were in close agreement; however, the P-retention by the fish (determined 

from body P content) was much lower than the digestibility or solubility data even in the group of fish fed a 

P-deficient diet. The authors explained, "In this experiment, 61.6-70.5% of absorbed P was retained and the rest of 

29.5 to 38.4% was probably returned into the digestive tract as observed in mammals or excreted through gills and 

urine. . . . Furthermore, experiments will be needed to clarify the destiny of absorbed P in fish." The "absorbed P" 

may include absorbed P by the fish, dissolved dietary P that was lost while the small carp was chewing the pellets, 

and any soluble P in feces that was lost before fecal collection. The excretion of endogenous (absorbed) P back 

into the digestive tract may not be a significant source of error in fish (thus, apparent availability of certain P 

compounds can be close to 100% in fish even at high dietary levels). The apparent digestibility, which includes 

endogenous P, does not explain the difference. Also, P-deficient fish do not excrete 33% of absorbed P via gills 

and urine. Spencer et al. (2000) estimated bioavailability of P in ordinary and low-phytate corn based on peptic 

and pancreatin digestion methods in vitro, and obtained similar values to those determined in vivo. 


41

The particle size 

Cromwell (1989) suggested that variable availability of P in meat and bone meal among investigators is at least 

partly due to differences in the bone particle size, and that large bone particles are poorly available to pigs. The 

author also stated that the availability of P in defluorinated rock phosphate is related to the particle size, with finer 

particles being slightly more available than coarse particles in pigs. Eya & Lovell (1997) reported that finely 

ground defluorinated rock phosphate was more available to channel cat fish than the coasely ground material (82% 

vs 55%). Vielma et al. (1999) reported that P in finely ground bone was more available for rainbow trout (initial 

wt 3 g) than P in coarsely ground bone based on whole-body ash, P and Ca concentrations. 

Measuring the availability of various P compounds 

VonGohren (1861) found that supplementing hay with Ca-Mg-phosphate greatly increas ed the retention of these 

minerals by the lamb. Soxhlet (1878) determined the retention percentages of ash, P, Ca, Mg, Fe, K, Na and Cl in 

milk by young calves. The data showed that 72.5% of P and 97% of Ca in the milk were retained. Tereg & 

Arnold (1883) measured the contents of P, Ca, and N in food, urine and feces of the dog fed diets supplemented 

with either Ca-carbonate, primary, secondary or tertiary phosphates, and estimated P retention based on the balance. 

Kohler et al. (1904) determined the retention of various P compounds in yearling lambs as follows: 13.1% for 

precipitated bone earth, 14.2% for calcined bones, 26% for dicalcic P, and 35.5% for tricalcic P. With a younger 

lamb, these P compounds were better ret ained. Higgins & Sheard (1933) found no differences in the assimilation 

of secondary and tertiary phosphates by the chick. Rottensten & Maynard (1934) compared the assimilation of P 

from dicalcium phosphate, tricalcium phosphate, bone calcium phosphate, and cooked bone meal, and found no 

significant di fferences among these sources. Gillis et al. (1948) determined P availability of various P sources in 

chicks. 

Uncommon P sources 

Paquelin & Joly (1877, 1878) administered 2 g of sodium pyrophosphate, Na 4P 2O 7, daily for 5 days or total 5 g of 

sodium hypophosphite, NaH 2PO 2, in 5 days to a woman, and found that both were apparently all eliminated in the 

urine unchanged. Panzer (1902) wrote, "Ca hypophosphites fed to a dog is quickly and almost completely 

absorbed, passes through the organism without being held back anywhere, and is completely eliminated within 24 

h." Heffter (1903) report ed, “in healthy organism phosphorus acid (H 3PO 3) is completely oxidized, 

metaphosphates, Na 3P 3O 9, are changed to the ortho-form, while pyrophosphates and hypophosphites are excreted 

unchanged.” Shelling (1932) studied the utilization of pyrophosphate, metaphosphate, and hypophosphite in 

parathyroidectomized rats, and reported that pyrophosphate was utilized while the latter two types were inert. 

Gillis et al. (1954) determined the relative bioavailability of 38 P compounds in chicks based on the slope ratio assay 

of bone ash with tricalcium phosphate as a standard. They reported that orthophosphates including bones, feed 

grade phosphates and defluorinated phosphates were more available (80% min) than pyrophosphates and 

metaphosphates. Agricultural grade phosphates (super phosphate, triple super phosphate, monoammonium 

phosphate) had comparable bioavailability to corresponding feed grade phosphates. Cromwell (1989) wrote that 

the availability of P in Curacao phosphate, soft phosphate, colloidal clay, and high-fluorine rock phosphate was quite 

low. Fernandes et al. (1999) fed chicks with a corn-soybean based diet supplemented with a test phosphate or 

purified dicalcium phosphate (standard), and determined the relative bioavailability by the slope ratio procedure 

based on weight gain, bone ash and bone strength. They noted that thermomagnesium appeared to be toxic, and 

that agricultural-grade phosphates contained fluoride in the amounts higher than feed-grade phosphates (0.4-1.1% vs 

0.03-0.2%), but the P in both sources was highly available. The authors suggested possible fluorine toxicity by 

feeding agricultural grade phosphate for an ext ended period. 

Measuring mineral absorption at various sections of the GI tract using an indigestible indicator 

Wildt (1874, 1879) conducted digestion experiments on sheep. He analyzed the contents of different parts of the 

alimentary tract for P, Ca, Mg, Na, K and Cl, and estimated their absorption during their passage through the 

alimentary tract. He used silica as an inert dietary indicator to trace their absorption. The data showed 

considerabl e secretion of (endogenous) minerals in certain parts of the digestive tract. This observation convinced 


42

many subsequent workers that measuring the apparent digestibility of mineral elements has little meaning in 

ruminant nutrition (see also “Endogenous P in feces” section). It should be noted that the same method is used 

today to measure nutrient digestibility. 

Spallanzani's digestion experiments 

--------- In preparation. 

Endogenous P in feces 

Jordan, Hart & Patten (1906) based on available evidence and their own experience with cows concluded that the 

percentage of digestibility of mineral compounds (including P) is not measured by the difference between the 

amounts of these compounds in the food and in the feces. Forbes & Keith (1914) also wrote, "Utter absurdity of 

the usual statements as to digestibility of mineral nutrients, and also of many statements as to coeffi cients of 

absorption of inorganic salts (pp.184)." and further "To relate the feces P compounds as a whole directly to those of 

the food, as in computation of digestibility, is without justification since a considerabl e part of the feces P have an 

origin other than in the food (pp.198)." Bondi (1987; pp. 175, 293) also wrote, "Digestibility coeffi cients are not 

calculat ed for minerals . . . values of digestibility coeffi cients of minerals would be meaningless." Similar 

statements are found in every textbook or article on mineral nutrition (e.g., Maynard & Loosli 1962; pp.129, 302, 

McDonald et al. 1988; pp. 207, 214, Lall 1979). 

It is generally agreed that the important measurement for minerals is true digestibility, which requires 

distinction between the portion in the feces which represents unabsorbed material (dietary) and that which repres ents 

discharged in the gut (endogenous). Gregersen (1911) reported that the daily elimination of total P of eight rats 

fed a N-free-P-free diet ranged 28-55 mg per kg of body weight, and that Ca and Mg tend to deflect P excretion into 

the feces. Nicolaysen (1937) fed rats a P-free diet to determine endogenous fecal P under normal and vitamin D 

deficient state. The author showed that vitamin D deficient rats excreted more endogenous P in feces than normal 

rats, and that the endogenous fecal P excretion increased as the dietary Ca increas ed. Kleiber et al. (1951) 

criticized feeding of a P-free diet for it introduces an "abnormal condition". They injected 32 P into a cow and 

showed that 43% of the total fecal P was endogenous. What the authors called endogenous loss was evidently the 

total of the absorbed P that was excreted into the gut (due to excess intake) and the obligatory loss, especially the 

former. Thus, the value can be different depending on the dietary P level. It should also be noted that feces is the 

principal path of P excretion in herbivore, but the urine is the principal path in carnivore, and the output may be 

divided between the two channels in the case of man depending on the Ca content in the diet (Liebig 1843; Paton et 

al. 1899-1900; Bergmann 1901; Sherman 1919, Maynard & Loosli 1962; Bondi 1987). 

Thompson (1965) discussed about how to deal with endogenous excretons of P and Ca in determining their 

true availabilities. As mentioned above, however, the endogenous fecal P is relatively small in carnivores--- this 

means that the difference between the apparent and true digestibility is small. If endogenous excretion represents a 

significant portion of fecal P, the apparent absorption of P as measured by fecal analysis cannot be very high. The 

apparent P absorption of highly available P sources has been report ed to be close to 100% in salmonid fishes (e.g., 

Ogino et al. 1979; Baeverfjord et al. 1998) and carp (Nakamura 1982), meaning that the endogenous excretion of 

P is practically negligible. Although the true digestibility is more meaningful than the apparent digestibility, the 

latter can still offer useful information. Firstly, the true digestibility is always higher than the apparent digestibility 

(useful fact to remember). Therefore, when apparent digestibility is 90%, true digestibility is between 90 and 

100%. Secondly, when comparing two or more P sources in a single feeding trial, a compound (or ingredient) of 

higher apparent P digestibility is also higher in true P digestibility than that of lower apparent P digestibility. If a 

standard such as KH 2PO 4 is measured each time for apparent P digestibility along with test sources, P availability 

may be expressed as relative to the standard in a similar way when calculating relative bioavailability based on bone 

calci fication. 

An important issue is that even if true availability of P (and other minerals, especially trace minerals) is 

determined precisely, the values cannot be absolute as McDonald et al. (1988) wrote, "The availability of minerals 

in a particular food depends so much on the other constituents of the diet and on the animal to which it is given, and 

therefore average values for individual foods would be of little significance." This may be the primary reason why 

no attempt has been made in the tables of food composition to give availability coeffi cients of minerals comparabl e 

to digestibility coefficients of organic nutrients. Mellanby (1925) mentioned long ago, ". . . the amount of Ca and 

P in the food is of but secondary importance in the control of the deposition of these elements in growing bone. In 


43

view of the evidence of interaction and balance among food constituents, . . . optimum Ca and P contents in diet 

vary every time the other elements of diet are changed." Sugiura et al. (1998) calculated the interaction 

(inhibition) potential of each feed ingredient by measuring mineral absorption when a semi-purified bas al diet was 

fed alone and when it was mixed with one of test ingredients; thus the digestibility of minerals can be negative due 

to the presence of interfering compounds in each feed ingredient. 

Fecal endogenous losses may be estimated using a linear regression of the excretion (or ret ention) on the 

intake, which is equal to the constant of the regression. Kienzle et al. (1998) estimated dietary P requirement for 

adult cats based on a factorial procedure. The authors determined endogenous fecal loss, endogenous renal 

excretion and true digestibility of P. A basal diet contained turkey meat (60%) as the major P source, which was 

supplemented with P (source unknown) with varied amount of Ca (Ca/P ratios from 1/1-4/1). This procedure, 

however, does not estimate the endogenous fecal P excretion accurately. When endogenous fecal P is to be 

determined as the constant of a linear regression line, the P availability (absorption) of the diet has to be constant 

throughout, i.e., constant regression coefficient. 

True digestibility by differential approaches 

The true digestibility of P may be determined without knowing the endogenous excretion. Steggerda & Mitchell 

(1939) determined the true availability of Ca in human diets by giving the test materials at two levels of intake, one 

very low and the other at about maintenance, and calculating the change in Ca retention relative to the change in Ca 

intake (i.e., True availability = ∆Retention/∆Intake). A similar approach has been reported by Ammerman et al. 

(1957) who determined the true absorption percentages of various P compounds by using the following equation; 

True absorption (%) = {(Total P intake – P in basal ration) – (Total P exc. – Basal P exc.)}*100/(Total P intake – P in 

basal ration). This formula may be simplified as True absorption = ∆Absorption/∆Intake. In this experiment, all 

lambs received a basal ration containing 0.032%P for 4 weeks, and the fecal collections of 5-7d-duration were made 

during the latter part of the 4wk-depletion period. The supplemental feeding of P lasted for 12d during which four 

3-day collections were made. The supplemental rations contained 0.154-0.158%P, which are well below the 

dietary requirement. Kleiber (1975) reported an equation to calculate "partial availability" as follows; Partial 

availability = (∆Intake – ∆Feces)/ ∆Intake, which is the same as Ammerman's calculation. Hurwitz (1964) 

reported a concept of "net P utilization". This method is also similar to the previously reported in that the Net P 

utilization =(∆P body/∆P intake)*100 The slope of abscissa of the dose-response curve determined on various P 

intakes will be (dP body/dP intake)*100. The author estimated total body P content from the tibia P-total body P 

relationship that had been determined separately. The slope was determined in the liniar regression below the 

minimum requirement. It should be noted that the term "net" normally indicates "apparent", but here the author 

used the term as "true". Hurwitz et al. (1978) calculated "fractional absorption" to determine % absorption of a P 

supplement at various Ca levels. The calculation is the same as Ammerman et al. (1957). Bondi (1987) reported 

a "comparative bal ance technique" to determine percentage of utilization of a nutrient. The calculation is as 

follows: Utilization (%) =∆Balance*100/∆Intake. Oldham & Emmans (1988) briefly discussed about absolute 

response (= output/input), incremental response (=∆output/∆Input), marginal effici ency (= slope of the regression 

line), and the differences between diminishing returns and the broken stick (line) responses. The differential 

method of estimating the true digestibility or availability is based on the assumption that the endogenous P excretion 

is constant at different dietary P intakes, the assumption of which has been questioned by Kleiber et al. (1951) as 

mentioned above. Ammerman (1995) also discussed this point by using terms "Minimum endogenous" 

(obligatory) and "Variabl e endogenous". When ∆intake is very small (i.e., similar levels of intake), the total 

endogenous will be similar. Thus, the slope method reported by Kleiber and Hurwitz as discussed above is free 

from such a compromise, but construction of a best-fit dose-response curve by measuring responses at various P 

levels will be essential (thus more laborious than the differential method). The slope method also gives the 

availability of a dietary nutrient at various fractions of dietary levels as reported by Gahl et al. (1995, 1996) for 

lysine using pigs and rats. The authors defined that the marginal efficiency (d retention/d intake) is the effici ency 

of retention or gain for a small increment of the amino acid added to the diet. The maximum marginal effici ency 

occurred at about 40% of the maximum retention of lysine. They concluded that since diminishing returns affect 

the upper 55-65% of the response range, this information has to be considered in formulating economically feasible 

diets that would maximize economic returns rather than maximize animal growth. Rodehutscord et al. (2000) 

reported similar results on P using rainbow trout. 


44

Errors due to high dietary P levels 

When estimating P bioavailability based on general responses such as wt gain, maximum P retention, and bone 

mineralization/strength, the content of available P in the diet should not exceed the dietary requi rement. Any 

excess portions of available P in the diet will be excreted by the animal, which therefore will be a direct source of 

error. Many biological measurements, including growth and bone mineralization, follow typical dose-respons e 

relationships (e.g., sigmoidal curve, polynomial regression curve). This means that the responses start to decreas e 

before the dietary requirem ent is met, and plateaued at or near the the dietary requirement. The decreased 

biological responses near or above the dietary requirement is known as "diminishing returns". Therefore, the 

response is normally measured where the dietary level is low enough so that the response is linear (maximum 

biological efficiency) to the dietary intake. However, in practical feeding, animals are almost certainly fed 

nutrients at least the requirement levels where the response is known to be much lower than the maximum response, 

raising some concern of using data determined under such an abnormal or extreme condition. 

When estimating P bioavailability based on absorption (digestibility), an excess portion of available P in 

thea diet will become a problem if the intestinal P absorption decreases or endogenous fecal P excretion increases at 

higher levels of intake. In ruminant animals, this is the case and the digestibility may not be known by fecal 

analysis (discussed in section “ Endogenous P in feces”). In humans, the gastrointestinal absorption of P is 

unregulated under normal conditions, and net P absorption is directly proportional to the amount ingested 

(Anderson 1991, Dennis 1992). IOM (1998) also wrote, “ there is as yet no evidence that the gastrointestinal 

absorption of P is regulated.” Any excess P that is absorbed from the diet will be excreted in urine, suggesting that 

the kidney is the site for P homeostasis. Brickman et al. (1974) noted that even in the face of dangerous 

hyperphosphatemia, P continues to be absorbed from the diet at an effi ciency only slightly lower than normal. 

However, Bachmann et al. (1940) showed that the rat fed casein-based semi-puri fied diet for 70 days had a lower 

percentage of P absorption when the dietary P level was high than when the dietary P level was low. The authors 

maintained the Ca/P ratio in the diets constant (1:0.57) by using CaCO 3 and H 3PO 4 as the Ca and P sources, 

respectively. If the dietary P level was increased without increasing the Ca level or if the feeding duration was 

short, the result might have been different. Hurwitz et al. (1978) reported that P absorption in turkey increased 

linearly with P intake, within each level of dietary Ca. They suggested that the lack of adaptation of the P 

absorption to either low or high intakes of P is a clear contrast to Ca absorption for which the intestine is the 

important site of Ca homeostasis. The authors wrote, "Since absorption continued to increase linearly with P intake 

and retention plateaued early in the curve, it is not surprising to find that urinary P increased as P absorption 

increas ed." Ammerman et al. (1960) determined P availability in chicks in 3-6 days following a 4 or 7 day 

depletion period. The bone ash % was linear up to 0.6%P when repletion (test) period was 3d, while it was linear 

up to only 0.4%P when the repletion period was 6d, suggesting that the test period and the diet history (body pool 

size of the test animal) are critical in studying biological responses. Ammerman (1995) also says that if feeding 

duration is relatively short, excess dietary intakes of minerals such as copper and manganese, even at near toxic 

levels, will not be a major problem in estimating the relative bioavailability based on their accumulation in specific 

tissues. This suggests that their absorption is unaffected by the levels of intake within a certain duration of feeding. 

According to Mertz (1987), balance studies can be used to compare the biological availability of different mineral 

sources, but such studies should be of short duration and should be terminated before a bal ance is established for 

substances both of high and low bioavailability. 

In carp, Nakamura (1982) reported that the apparent absorption of P was unchanged (~90%) regardl ess 

of the P content in diet (0.35-3.14% P) when Ca content in the diet was kept constant at 0.7%. Thus, the amount of 

P absorbed from the diet was directly proportional (linear) to the amount of P in the diet. Satoh et al. (1992), 

however, found that rainbow trout absorbed 60% of P in a fish meal-based diet; but, when the diet was supplemented 

with water-soluble P in an amount corresponding to the dietary requirement, the fish did not absorb P from the fish 

meal. Yet, Satoh et al. (1996) reported that rainbow trout did not regulate the absorption of P when a casein-based 

semi-puri fied diet (of low Ca content) was supplemented with monopotassium phosphate to contain 1.2-3.0% total P 

in the diet. The apparent absorption was 85% (when diet contained 1.2%P) and 94% (when diet contained 3.0%P). 

Satoh et al. (1997), using egg albumin-based purifi ed diet and monocalcium phosphate as a P source, noted that the 

absorption of P in carp was not influenced by the dietary available P level, whereas, in rainbow trout, the amount of 

absorption continued to increase but with decreasing percentages when the diet contained P in amounts two or three 

times higher than the dietary requirement. In pigs, however, Schroder, Breves & Rodehutscord (1996) and 

Rodehutscord et al. (1998) are critical about the upper level of dietary P, which, they say, must be suboptimal 

(below the net requirement) when determining digestibility (absorption) of dietary P. In rainbow trout, 

Rodehutscord et al. (2000) determined the marginal P absorption (= ∆apparently absorbed/ ∆intake) and marginal 

efficiency of P utilization (= ∆retained/ ∆intake) at various dietary P levels, which showed the diminishing returns 


45

(i.e., decreasing P utilization efficiency by increasing dietary P intakes). They suggested that the upper limit of 

available P in diet, when determining P availability based on apparent absorption, should not exceed 0.25%. This 

is only 40-50% of the dietary requirement for the fish. Data obtained under such an extreme condition may 

probably need some justification, if they are to be used for normal fish that are receiving and must receive at least 

the requirement level of P in diet under normal aquaculture feeding. 

Phase feeding & finishing feed 

The aims of phase feeding are to minimize the feed cost and waste excretion by tailoring nutrient contents in diets to 

the speci fic requirements of the animals of di fferent li fe stages, physiological conditions (e.g., reproductive, laying, 

spawning stages) and breeding conditions. Finishing feed is one of the phase feeds, but is used specifically before 

harvesting, to alter or modify the final product quality (in addition to the above-mentioned aims). Phase-feeds and 

finishing feeds are common in pig and poultry industries; however, they are less common in fish feeds. The reason 

for that is that fish continue to grow at least until the marketing sizes, and that the nutrient requirements may not 

change so greatly as other species (e.g., baby to adult pigs or broiler to laying hens). Another important reason is 

that there is little data about nutrient requirements speci fic for large fish (from grow-out to harvesting sizes)--- see 

section “P requirement for large fish”. 

Hardy et al. (1993) studied the effect of phase feeding, and noted that dietary P levels did not alter apparent 

P absorption or availability in juvenile rainbow trout, and that P-deficient fish did not show compensatory increase 

of P absorption. The compensatory increas e of digestive or absorptive capacity seems to be limited in fish since 

most P in fish diets is absorbed passively, which is not regulated (cf. chapter “P-transport”). The authors also 

found that, as far as fish have P reserve in the bones, the fish can grow normally until the P store is used up beyond a 

certain threshold level. Thus, feeding P-defi cient diets for a certain period (depending on the degree of P 

deficiency of the diet and the initial P reserve in fish body) is possible without sacrificing or compromizing fish 

growth (see section “ Critical factor: Growth magni fication”). Lellis et al. (2004) recently confirmed thes e findings, 

and reported that dietary P level could be reduced to as low as 0.15% when the intial fish size was 400 g and the 

harvesting size was set at 550 g. When the initial size was 200 g and 300 g, the diet needed to contain minimum 

0.6% and 0.3%P, respectively to maintain optimal fish growth and product quality. As the authors suggested, the 

body P reserve of fish differs depending on the fish size and previous diet history, and thus examing the body P 

status or reserve of the population is essential before starting P-deficient regimen to minimize the risk of clinical P 

deficiency. Because currently the precise dietary P requirement for large fish is still uncertain, especially when 

considering the nature of dietary requirement that varies greatly depending on the basal diet, the use of moderately P 

deficient finishing diets should be considered the best available option in environmentally friendly feeding. In this 

regimen, however, periodic monitoring of fish P status will be essential to avoid the clinical P deficiency. 

Another benefit of using P-defici ent finishing diet is to modify the final product quality. As is clear from 

Table 2, P-defici ency increases body fat deposition in the fish. By feeding P-defici ent finishing diet, it is possible 

to make fatty fish--- which have higher commercial value than lean fish in certain cas es and countries. Also, it 

may be effective to use P-deficient finishing diets to alter body fatty acid profile of the fish (to increase shel f li fe, 

omega-3, etc) since the catabolism of dietary fats seems to be blocked in P-defi ciency (thus, effectively stored in the 

body). This means that the fillet texture, flavor, frozen stability, nutritive value and among other quelities can be 

improved--- by using P-defici ent finishing diets. 

Relative bioavailability 

Gillis et al. (1954) and Nelson & Walker (1964) used the slope-ratio assay on bone ash (%) to determine the 

relative bioavailability of numerous P compounds for the chick. Lofgreen (1960) using a radioisotope dilution 

technique determined the true digestibility of several inorganic P supplements for sheep. The true digestibility of P 

in dicalcium phosphate was determined to be 50%, whereas its apparent digestibility was only 10%. Using this 

data, Peeler (1972) assigned 100% biological value on dicalcium phosphate, and expressed the data on a relative 

scale instead of digestibility. The relative bioavailability of P, therefore, became two times higher than the true 

digestibility. Sauveur & Perez (1987) showed the difference between relative bioavailability and true digestibility 

(absorption) using pig data. The relative bioavailability of P in such compounds as monocalcium phosphate, 

monosodium phosphate and phosphoric acid was 100 (often used as the reference); however, the true digestibility of 

those sources was only about 60-80%. The relative bioavailability is also variable depending on the reference used. 

In any case, the true availability (or true digestibility) of the standard compounds need to be known, so that the true 


46

availability of the test compounds may be calculated from the relative value determined. Rodehutscord (1995) 

concluded, based on several balance trials, that digestible P is completely available in the pig's intermediary 

metabolism. Bone ash percentage and bone-breaking strength are the most common dependent variables in 

determining the relative P bioavailability (Soares 1995); however, numerous other criteria have also been used 

including growth rate, feed consumption, feed effici ency, skeletal abnormalities, net P retention, blood phosphatase 

activity, rate of absorption, P balance, blood serum P levels, and others (Peeler 1972). In addition, Sullivan (1966) 

calculat ed the relative bioavailability of P for turkeys based on combined responses of weight gain, feed effi ciency 

and bone ash instead of using only one criterion. Other factors such as the duration of feeding and the size of 

animals also likely affect the measurement. Cromwell (1989) and Littell et al. (1995) discussed methods of 

determining relative bioavailability. There are some review articles on P availability of various sources in various 

animal species (see Nelson 1964, Soares 1995, NRC 1998) and fish (NRC 1993). 

P availability in Carp 

In carp, Viola et al. (1986a) reported that the availability of P in fish meals and seed grains was very low, and that of 

dicalcium phosphate was high. Huang & Liu (1990) reported that P digestibility of inorganic P supplements 

determined in grass carp was similar to those in carp, but P digestibility of natural feed ingredients was higher in 

grass carp than in carp (see Table 1). 

P availability in Salmonids 

Ogino et al. (1979) determined P availability of various feed ingredients and P supplements for carp and rainbow 

trout based on apparent absorption (see Table 1). Fish growth, feed effi ciency, proximate body composition, 

vertebral Ca and P contents and vertebral abnormality of the fish were also studied. Riche & Brown (1996) 

determined in rainbow trout the apparent and true availabilities of P in several feed ingredients by fecal collection 

(by dissection). Their true availability values seem to agree with the apparent availability values reported by other 

workers, but their apparent availability values are unique. What the authors called "endogenous P" is apparently 

the total of endogenous P and undigested dietary P, especially the latter. They used barium carbonate as an "inert 

indicator" to determine P absorption. Barium carbonate is highly toxic and is used as a rat poison (Merck Index, 

1983; NRC, 1980). Riche et al. (1995) used barium carbonate at a 0.5% dietary level, but at a 1% level the fish 

did not eat. Nordrum et al. (1997) determined P availability in various inorganic sources and bone meal for 

Atlantic salmon based on retention (values are shown in Table 1). The fish grew only from 5.6 to ca.10 g in 12 

weeks of satiation feeding (fed every 15 min) with automatic feeder. The feed effici ency was fairly good (ca. 1.3), 

suggesting that the feed intake was very low. The casein-gelatin semi-puri fied diet (basal diet) to which one of test 

P sources was supplemented (test diets) contained 0.46-1.10%P, which appears to be higher than the amount that fish 

can retain. The authors attempted to equalize the Ca/P ratio of all diets. Thus, the basal diet was very different in 

calcium content from one diet to another. This annulled the constant P availability of the basal diet (the assumption 

to calculate digestibility). Rodehutscord et al. (2000) determined marginal and overall P digestibility of Na 2HPO 4 

(feed grade) at various dietary P levels in rainbow trout. The maximum P digestibility of the P source was only 

75%. Johnson & Summerfelt (2000) used blood meal at a level of 8.8% in the diet for rainbow trout, which 

replaced a portion of herring meal, while maintaining protein and caloric content of both diets constant. The use of 

blood meal did not reduce the growth rate of trout, but reduced P excretion by the fish at the level of 22.7% 

compared with those fed fish meal diet. Vielma et al. (2000) fed rainbow trout (initial body wt. 0.25 kg) for 6 

months with either fishmeal diet or fishmeal -soyprotein diet. The fish grew up to 2.02 kg (average final wt.). The 

weight gain and feed efficiency did not differ, whereas P excretion was markedly low in fish fed fishmeal-soyprotein 

diet (4.6 g P/kg wt. gain) compared with those fed fishmeal diet (8.5 g P/kg wt. gain). 

P availability in Catfish 

Lovell (1978) determined the apparent availability of P for various feed ingredients and P supplements using 

channel cat fish (body wt 280g), chromic oxide as an indicator, and intestinal dissection to collect feces (data given 

in Table 1). The author raised a question about the high value of measured P availability of soybean meal 

(50-54%) bas ed on non-phytate P content in soybean meal, which is normally one-thirds of the total P. Wilson et 

al. (1982) reexamined the apparent P availability of soybean meal in channel cat fish (0.5-1.0 kg body wt). The 

values they obtained ranged from 27 to 32%. They also determined the apparent P availability of casein (90%) and 


47

egg albumin (71%). However, the diets were supplemented with Ca carbonate in amounts of 0.5% and 1.18%, 

respectively. The diets therefore had high Ca/P ratios (especially egg albumin diet), which likely decreas ed P 

absorption or availability. The large difference in estimated P availability of soybean meal between the two studies 

may be due to different Ca contents of the diets since free phytate (phytic acid) and calcium-phytat e (phytin) are 

different in bioavailability. Since Bruce & Callow (1934), many workers reported in various species that Ca 

interferes with the utilization of phytate P by forming Ca-phytate precipitate in the intestine. If phytate stays in the 

soluble fraction in the intestine, it can be digested by bacterial, dietary, and/or endogenous phytases. Apparently, 

the diet used by Lovell (1978) contained little Ca since this researcher used a Ca-P-free mineral mixture, whereas 

Wilson et al. (1982) used a diet containing a substantial amount of Ca as Ca carbonate and Ca lactate, which was (in 

my calculation) capable to precipitate as much as 0.47% phytate-P in the diet. Taylor (1965) wrote, "Phytate-P is 

by no means completely undigested in the rat, dog, pig and hen under appropriate conditions, and it appears that the 

extent of phytate breakdown in the gut is reduced as the level of dietary Ca increases." (see section “Precipitation 

of calcium phytate”) It should be noted that about 60% of phytate in soybean meal are water-soluble and easily 

removed by washing (Han 1988). Any portion of dietary phytate that leaches out from the pellets before being 

ingested by the fish therefore will be calculated as "digestible". Also, the leaching loss of phytate from feces will 

be accounted for as "digested". The degree of thes e sources of error depends on water stability of the pellets, fish 

size (pellet size), palatability, feeding level, feeding method, fecal collection method, and the content of cations in 

the diet. Both Lovell (1978) and Wilson et al. (1982) collected fecal samples by dissection; however, the former 

apparently fed the diet manually, while the latter force-fed the diet. Li et al. (1996) compared bioavailability of P 

in feed-grade dicalcium phosphate and 3 defluorinated rock phosphates of high, midium and low solubilities in 

neutral ammonium citrate. Channel catfish (body wt. ca.4 g) were fed in aquaria for 12 weeks with egg 

albumin-based puri fied diets containing one of the test P sources. Based on weight gain, feed consumption, feed 

conversion ratio, bone ash and P levels, they reported that all the test P sources had comparable bioavailability for 

channel cat fish. Eya & Lovell (1997) determined P availability of various P supplements for channel cat fish based 

on absorption (digestion). They used a non-purified diet containing soybean meal as the basal diet rather than 

using a purified low-P diet. The authors subtracted P absorbed from the basal diet portion of the test diet to 

determine the amount of P absorbed from the test P source. This method is acceptable i f interaction is negligible. 

The calculated values are the true absorption (availability) although the authors called the values net absorption 

(which normally refers to as apparent absorption). The authors added CaCO 3 to maintain 1/1 Ca/P ratio in all diets. 

The values could have been higher if the diets were low in Ca. 

P availability in Tilapia 

Viola et al. (1986b) reported, in tilapia, that P in various fish meals was highly available, soybean P was poorly 

available, and sorghum P was superior to wheat P. They noted that fast growing fish need more P than slow 

growing fish. 

P availability in Other fishes 

In red sea bream, Sakamoto & Yone (1979) reported that sodium phosphates (mono-, di-, and tribasic), potassium 

phosphate monobasic, and Ca phosphate monobasic were more effective than Ca phosphates (di- and tribasic) to 

prevent the development of P deficiency. Ca phytate was poorly utilized by the fish as a P source. Yone & 

Toshima (1979) showed that the digestibility of P in fish meal was higher in seabream than in carp. Carp fed fish 

meal diet grew poorly with very low feed effi ciency, which was improved by supplementing the diet with available P. 

Fernandez et al. (1996) studied the absorption of C, N, P and dry matter along the intestine of gilthead seabream, 

and compared di fferent fecal collection methods to estimate apparent digestibility coeffi cients. Fernandez et al. 

(1998) determined again the apparent digestibility from several regions of the intestine of gilthead seabream (body 

wt 10-25 g). Except for P, the apparent absorption increased along the intestine. High correlations were found 

between N, C and DM digestibilities, whereas P digestibility showed low or no correlation. Silva & Oliva-Teles 

(1998) determined the apparent digestibility coefficients of dry matter, protein, energy and P in two fish meals, a fish 

protein hydrolysate, blood meal, meat meal, soybean meal and yellow dextrin using seabass (body wt 40 g). The 

optimal inclusion level of a test feedstuff into the reference diet in estimating the apparent digestibility was also 

studied (15 and 30%). Satoh et al. (1998) reported that fish meal-bas ed diet containing total P at the level of 2.1% 

did not provide optimum growth of red seabream (body wt., initial 2.4 g, final 23 g vs. 30 g), and concluded that 

supplementing the diet with available P was necessary. The fish fed the low-P diet, however, had similar or higher 


48

P contents in the whole body and vertebrae than those fed P-forti fied diets. 

Conclusion 

Aquaculture as well as other food production systems pollute aquatic environment by the discharge of effluent water 

high in phosphorus, nitrogenous compounds, and organic matter. As a result of increasing environmental concern, 

regulatory pressure to aquaculture industry increased, which produced both solutions and problems. Numerous 

benefits of aquaculture have been reduced under the current legislature. In the US, aquaculture benefits human 

nutrition by supplying omega3 fatty acids (EPA, DHA) that reduce the risks of heart diseases, stroke and obesity, 

and enhance brain development of children. In developing countries, small-scale aquaculture prevents malnutrition, 

and contributes to poverty alleviation with minimal risk of entitlement failure. Aquaculture is the only hope to 

compensate decreasing supplies of fish from traditional capture fisheries. Developing technologies that alleviate 

environmental pollution, however, received less attention than establishing environmental regulations. In order to 

take advantage of the above-mentioned benefits of aquaculture, it is critical to support aquaculture development. 

The ultimate source of pollution in aquaculture is feeds. Nutrients in the feeds that are not digested or absorbed or 

in excess of requi red by fish will be excreted into water. Solid wastes may be collectable by settling, whereas 

soluble components are discharged into rivers, lakes, and coastal areas. Effluent phosphorus excretion must be 

reduced to the minimum without the risk of phosphorus deficiency epidemic in cultured fish. As research data and 

findings compiled in this review demonstrate, reducing phosphorus pollution associated with aquaculture is 

achievable by research and technology development, rather than laws and policies. Prudent environmental 

decision-making must therefore include an effort for newer technologies in order to ensure sustainable development 

of aquaculture. 


49

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74

Table 1. Availability of Phosphorus in Various Feed Ingredients and S upplements 

Trout Catfish Carp Tilapi 

a 

Other fish spp. Other animal spp. 

Barley (whole grain) 47 2 , (K35,50) 13 , P28 13 , 

(P30) 13 , P45 25 , 

Blood meal 100 2 , A81 10 , (P92) 13 , 

Canola meal 48 9 , (P21) 13 , 

Casein 90 3 , 90 5 , 97 3 , A72* 7 , A92 10 Corn (whole grain) 36-37 

, 

2 , 25 4 , 34 29 , G71 26 , (K12,33,80) 13 , 

P12-48 13 , (P14,29) 13 , 

Corn gluten meal 2 1 , 9 2 , 44 9 , C16 2 , (P15) 14 Cottonseed meal 43 

, 

#11 , 

73 29 , 

D40 12 , (K42,80) 13 , (P1,42) 13 , 

Egg albumin 71 5 Feather meal 

, 

62-79 2 , C75 2 , A77 10 , (P31) 14 Fish meal (anchovy, 50 

, 

sardine) 

2 , 36-37 9 , 40 4 , C47 2 , G33 26 , 

Fish meal (herring, capelin) 44-52 2 , 55 9 , C57 2 , A52-53 10 Fish meal (menhaden) 35-37 

, 

2 , 22 9 , 39 4 , 46 29 , 

75 #11 C40 

, 

2 , A87 10 , 

D48 12 (K100) 

, 

13 , (P94) 14 , 

Fish meal (Peruvian) 44 2 Fish meal (brown fish 

, 

70,72,81 

meal) 

3 , 

54 20 13,25,3 

, 

3 3 , 1 20 Fish meal (whitefish) 37 

, 

1 , 36-47 2 , 

60,72 3 , 62 20 , 

10,18,2 

6 3 , 0 19 , 

4 20 , 

65 6 , C55 2 , A79 10 , 

T71 16 , B29 19 , 

Fish meal (whitefish, 12-17 

high-ash) 

2 , 

Fish meal (unspecified) K88* 13 , (K102) 13 , 

(P100) 13 , P85 25 , 

Lupin, extruded 62 28 , U100 28 Malt protein flour 36 

, 

1 , 

Meat & bone meal 22-27 2 , 53 29 , D66 12 , (K90,99,102) 13 , 

(P64,102) 13 , 

Meat & bone meal 

(low-ash) 

35 2 , 

Meat meal 3 2 Meat meal (low ash) 

, 

45 2 , 

Peas, extruded 43 28 , U100 28 , 

Peanut meal 42 9 , G70 26 , (P12) 13 , 

Poultry byproduct meal 48-64 2 , −8 9 , C68 2 , A81 10 , 

D27 12 , 

(K101) 13 , 

Poultry byproduct meal 

(low-ash) 

47-51 2 , 

Rapeseed meal (solv. ext.) 26 28 , U49 28 , 

Rapeseed meal (heated) 42 28 , U65 28 , 

Rice bran 19 3 , 51 29 , 25 3 , (P25) 13 Sorghum 36 

, 

29 , 

Soybean meal (solv. ext.) 14 1 , 22-27 2 , 

10-17 9 , 

50-54 4 , 

27-32 5 , 

49 #11 , 

36 29 C28 

, 

2 , A36 10 , 

D47 12 , G67 26 , 

(K40) 13 , P27 13 , 

(P23,31,36) 13 , P31,42 25 , 

Soybean meal (full fat) 32 9 , 


75

Soybean meal 

(de-phytinized) 

93 2 , 

Wheat 47 2 , 56 29 , C50 2 , D79 12 , (K38,43,58) 13 , P46 13 , 

(P49,51) 13 , P61-74 25 , 

Wheat middling 55 2 , 28 4 , 78 29 , 

38 #11 , 

C41 2 , A32 10 , 

G54 26 , 

(P41) 13 , 

Wheat bran (K23) 13 , (P29) 13 , 

Wheat germ 58 3 , 57 3 Wheat gluten meal 75 

, 

2 , C57 2 , 

Yeast (brewer’s, dried) 91 3 , 93 3 , A79 10 , 

Phosphoric acid 60,85,100 21 , 

80,94 22 , 

Monopotassium or 

Monosodium phosphate 

98 3 , 96,96 20 , 90 4 , 89 24 , 94 3 , 

98,98 20 , 

98,90 23 , 

S68 8 , A131* 7 , 

A94,95 10 , G90 26 , 

Monocalcium phosphate 94 3 , 94 20 , 94 4 , 94 3 , 

94 20 A86* 

, 

7 , S46 8 , 

A90 10 , G90 26 Monocalcium phosphate 

(feed-grade) 

81 

, 

24 , 

Disodium phosphate 100 18 , 94 20 , 

64-75 27 , 

97 20 , 

Dicalcium phosphate 71 3 , 65 4 , 

82 #11 , 

46 3 , A91* 7 , S19 8 , 

A72 10 , G60 26 , 


(K128) 13 , (P100) 13 , 

(K96) 13 , (P100) 14 , 

P96 25 , 

(K99,100) 13 , P91 25 , 

(P101,121) 13 , (P100) 14 , 

(K86-99) 13 , P32-71 13 , 

(P95,107) 13 , P70 13 , 

P87 25 , P62-75 25 , 

Dicalcium phosphate 

(feed-grade) 

75 24 , (K81-100) 13 , 

(K79,87,89) 15 , 

Tricalcium phosphate 64 3 , 64 20 , 13 3 , 

13 20 , 

S10 8 , A56 10 , 

G3 26 , 

(K93-100) 13 , 

Tricalcium phosphate 

55 

(feed-grade) 

24 , 

Soft rock phosphate (K38) 13 , P28,57 13 , 

Defluorinat ed phosphate 55-82 24 , (K69-96) 13 , P41 13 , 

P73 13 , (P87,100) 13 , 

(K82) 15 , 

Super phosphate 

(agri-grade) 

(K95) 13 , (K93) 15 , 

Triple super phosphate 

(K96) 

(agri-grade) 

15 , 

Monoammonium 

phosphate (feed-grade) 

85 24 , 

Monoammonium 

phosphate (agri-grade) 

(K100) 15 , 

Ammonium polyphosphate (K95,118) 13 , 

Bone meal (fish) A51* 7 Bone meal (unspeci fied) 

, 

(K94) 13 , (P82) 13 , 

P68-85 25 , 

Phytin 19 3 , 1 4 , 8,38 3 , A0 10 , (K0-60) 13 , (P25-40) 17 , 

Availability values are derived based either on the apparent absorption (no mark), or true absorption (values 

underlined), or retention (values with asterisk), or growth (values with # sign); or relative value (values in 

parenthes es; with bone ash or breaking strength as response criteria; standards used - varied). All values are from 

in vivo (feeding) trials. 

Species code: A (Atlantic salmon); C (coho salmon); D (red drum); G (grass carp); S (shrimp, prawn); T (chum 

salmon); B (seabream); U (turbot); K (chickens, turkeys); P (pigs); H (humans); R (rats). 

References: 1 Yamamoto et al. (1997); 2 Sugiura (1998); 3 Ogino et al. (1979), 4 Lovell (1978), 5 Wilson et al. 

76

(1982), 6 Watanabe et al. (1980a), 7 Nordrum et al. (1997), 8 Davis & Arnold (1994), 9 Riche & Brown (1996), 10 

Lall (1991), 11 Li & Robinson (1996), 12 Gaylord & Gatlin (1996), 13 Soares (1995), 14 NRC (1998), 15 

Fernandes et al. (1999), 16 Watanabe et al. (1980b), 17 Peeler (1972), 18 Rodehutscord (1996), 19 Yone & 

Toshima (1979), 20 Satoh et al. (1992), 21 Phillips et al. (1958); values at 4/1, 1/1, and 0/1 Ca/P ratios, 

respectively, 22 Phillips et al. (1959); values at 0.5/1-2.0/1 and 0/1 in Ca/P ratios, respectively, 23 Nakamura 

(1982), values at 0.1% and 0.7% Ca/diet, respectively, 24 Eya & Lovell (1997), 25 Schroder et al. (1996), 26 

Huang & Liu (1990), 27 Rodehutscord et al. (2000), 28 Burel et al. (2000), 29 Buyukates et al. (2000) 


77

Table 2. Deficiency S igns and Diagnostic Indicators for Phosphorus in Fish 

Criteria Response sensitivity 


High Low 

General 

Growth (weight gain) All but → 6, 7, 8, 14, 15, 33, 35, 

36, 47, 50, 

Protein (N) retention or excretion, PER 10, 19, 20, 21, 22, 53, 

Feed efficiency (feed conversion) 2, 3, 5, 9, 10, 11, 14, 16, 17, 

19, 21, 22, 23, 24, 25, 26, 29, 

30, 32, 36, 37, 38, 41, 42, 43, 

6, 8, 12, 14, 15, 20, 33, 

34, 35, 36, 50,52, 

Condition factor 

44, 46, 53, 54, 55, 

11, 2, 12, 25, 30, 

Dressing percentage 6, 

Hepatosomatic index 1, 12, 2, 11, 52, 

Intestine + adipose tissue / body 30, 2, 

Scale weight / body weight 35, 

Appetite (feed consumption) 8, 9, 24, 36, 55, 11, 12, 18, 19, 

Disease resistance, immune function 13, 51, 

Skeletal deformity 7, 16, 17, 18, 19, 26, 31, 36, 

43, 

Lethargy 8, 

Mortality, survival 5, 31, 34, 36, 3, 8, 14, 15, 17, 19, 20, 

21, 25, 35, 41, 53, 54, 

Reproductive performance (egg or larval quality, etc.) 27, 28, 

Weight loss on starvation 

Proximate 

30, 

Moisture, dry matter (body, liver, muscle) 3, 6, 21, 22, 1, 2, 11, 12, 16, 17, 19, 

20, 23, 27, 29, 30, 31, 

43, 52, 53, 

Protein (body, muscle, liver) 6, 12, 23, 1, 3, 9, 11, 16, 17, 20, 

21, 22, 27, 29, 38, 43, 

46, 50, 52, 53, 

Protein, albumin (blood plasma, serum) 11, 12, 20, 22, 

Protein digestibility 20, 55, 

Fat (body, liver, vertebrae, muscle) 1, 2, 6, 9, 11, 12, 16, (17), 19, 3, 11, 20, 23, 27, 50, 52, 

21, 22, 29, 30, 33, 43, 46, 49, 53, 

Fat (viscera) 6, (17), 19, 30, 43, 27, 

Fat deposition 22, 

Fat (% absorption, retention) 55, 2, 20, 

Non-polar lipids (liver, muscle, viscera) 2, 30, 

Polar lipids (liver, muscle, viscera) 2, 30, 

Energy (% retention) 9, 20, 

Sugar (starch) digestibility 

Inorganic 

55, 20, 

Bone breaking strength, density 6, 31, 

Ash, Ca, P (body) 3, 7, 8, 9, 16, 17, 22, 23, 26, 1, 14, 20, 23, 27, 34, 

29, 31, 32, 33, 38, 39, 43, 46, 

49, 50, 52, 54, 56, 

53, 

Ash, Ca, P (vertebrae) 1, 3, 4, 5, 6, 7, 11, 14, 15, 17, 11, 12, 14, 16, 23, 25, 

26, 31, 32, 35, 36, 37, 38, 41, 

43, 49, 50, 52, 54, 56, 

27, 34, 44, 

Ash, Ca, P (scales, skin with scales) 31, 32, 33, 35, 36, 54, 34, 44, 

Ash, Ca, P (liver) 11, 12, 20, 21, 27, 

78

Ash, Ca, P (muscle) 21, 31, 35, 

P, inorganic (blood plasma, serum) 1, 5, 6, 7, 9, 11, 12, 13, 22, 

24, 25, 34, 40, 42, 47, 49, 50, 

52, 55, 56, 

11, 36, 44, 

P, total (blood plasma, serum) 24, 31, 33, 

P (urine, nonfecal) 45, 50, 52, 55, 56, 

P (% absorption, retention, fecal excretion) 9, 14, 31, 55, 8, 20, 40, 53, 

Ca (blood plasma, serum) 5, 11, 11, 12, 22, 24, 25, 31, 

33, 34, 36, 40, 42, 44, 

50, 

Ca/P ratio (body) 3, 17, 22, 26, 

Ca/P ratio (vertebrae) 11, 1, 3, 17, 25, 26, 27, 35, 

43, 

Ca/P ratio (blood) 11, 12, 25, 11, 

Mg (body) 3, 7, 8, 9, 31, 33, 38, 17, 23, 49, 50, 

Mg (vertebrae) 3, 7, 23, 31, 35, 44, 11, 12, 17, 27, 52, 

Mg (blood plasma, serum) 44, 50, 22, 33, 

Mg (scales, scales+skin) 33, 35, 44, 

Zn (body, vertebrae) 11, 23, 49, 52, 7, 11, 12, 23, 50, 

Zn (plasma, serum) 20, 44, 50, 

Cu (vertebrae) 11, 52, 11, 12, 

Mn (body, vertebrae) 11, 50, 52, 11, 12, 23, 49, 

Fe (body, vertebrae, serum) 52, 11, 12, 22, 23, 

Mg, Zn, Cu, Mn, Fe (liver) 12, 

K (body, vertebrae, plasma) 9, 17, 50, 23, 38, 50, 52, 

Na (body, vertebrae, plasma) 

Biochemical & Physiological 

9, 17, 23, 38, 40, 50, 

52, 

Total cholesterol (blood plasma, serum) 11, 12, 2, 

Triglyceride (blood plasma, serum) 2, 12, 11, 

Phospholipid (blood plasma, serum) 2, 20, 

Free fatty acids (blood plasma, serum) 2, 

Fatty acid profile (muscle, liver, viscera) 30, 

Glucose (serum) 22, 

Glycogen, sugar (liver, body) 1, 20, 

Hematocrit, Hemoglobin (blood) 37, 42, 11, 12, 20, 24, 40, 

RBC, MCH, MCV, MCHC 20, 

ESR 42, 

Alkaline phosphatase (blood plasma, serum) 6, 7, 33, 

Alkaline phosphatase (intestine) 33, 

ATPase (myosin, actomyosin) 21, 

GOT, GPT (serum) 22, 

GOT, GPT, Glutamate dehydrogenase, CCE, PGDH, 

FDPase, PFK, PEPCK (liver) 

30, 

Pyruvate kinase (liver) 30, 

ATP, PCr, G6P, glucose (blood, skeletal muscle) 45, 

1,25(OH) 2D3, 25(OH)D3 (blood plasma, liver) 

Sodium-phosphate symporter (NaPi) protein or 

mRNA (kidney, intestine, pyloric caeca) 

48, 

48, 50, 

Pi uptake, active Pi transport (intestine) 47, 

 

These indicators, if sensitive, are also used as the response criteria for estimating dietary requirement of P in fish. 

Refer to each reference for the species studied. 

 

Numbers indicate the references as follows: 1 (Sakamoto & Yone 1978); 2 (Sakamoto & Yone 1980); 3 (Ogino & 


79

Takeda 1978); 4 (Lovell 1978); 5 (Wilson et al. 1982); 6 (Eya & Lovell 1997); 7 (Shearer & Hardy 1987); 8 

(Hardy et al. 1993); 9 (Rodehutscord 1996); 10 (Takeuchi et al. 1993); 11 (El-Zibdeh et al. 1995a); 12 (El-Zibdeh 

et al. 1995b); 13 (Eya & Lovell 1998); 14 (Ketola & Richmond 1994); 15 (Robinson et al. 1987); 16 (Shim & Ho 

1989); 17 (Watanabe et al. 1980b); 18 (Murakami 1967); 19 (Murakami 1969, 1970); 20 (Shimeno et al. 1994); 21 

(Takamatsu et al. 1975); 22 (Shitanda & Ukita 1979, Shitanda et al. 1979); 23 (Satoh et al. 1998); 24 (Sakamoto & 

Yone 1973), 25 (Yone & Toshima 1979); 26 (Ogino & Takeda 1976); 27 (Watanabe et al. 1984a, 1984b); 28 

(Watanabe et al. 1984c); 29 (Elangovan & Shim 1998); 30 (Takeuchi & Nakazoe 1981); 31 (Baeverfjord et al. 

1998); 32 (Schäfer et al. 1995), 33 (Skonberg et al. 1997), 34 (Brown et al. 1993), 35 (Davis & Robinson 1987), 

36 (Dougall et al. 1996), 37 (Andrews et al. 1973), 38 (Dove et al. 1976), 39 (Lovell & Li 1978), 40 (Nakamura 

1982), 41 (Li et al. 1996), 42 (Firdaus & Jafri 1996), 43 (Ogino et al. 1979), 44 (Vielma & Lall 1998b), 45 

(Sugiura 1998), 46 (Chavez-Sanchez et al. 2000), 47 (Avila et al. 2000), 48 (Coloso et al. 2003), 49 (Vielma et al. 

2002), 50 (Vielma et al. 1999), 51 (Jokinen et al. 2003), 52 (Roy & Lall 2003), 53 (Pimentel-Rodrigues & 

Oliva-Teles 2001), 54 (Borlongan & Satoh 2001), 55 (Rodehutscord et al. 2000), 56 (Bureau & Cho 1999), 

Classification between high and low sensitivities is arbitrary. Good reasons for the low responses might be 

suggested by the investigators themselves (short feeding duration, large fish size, enough dietary P, low feed 

efficiency, pond experiment, sampling time, methods, etc.), but they are not cited in this table. References that 

appear in both columns in the same row have conflicting results from multiple experiments in the same paper. 

Reference numbers in parentheses indicate that the response is opposite direction compared with the others. 


80

Table 3. Reference Values for Diagnosis of Phosphorus Status of Fish 

Variab 

le 

Part 

(wet or dry) 

Ash whole body, 

wet 


Normal range or ± SD (%) 

Trout Catfish Carp Tilapia Other spp. 

2.3-2.8 4 , 2.4-2.5 17 , 

2.4-2.6 19 , 2.4-2.8 39 , 

2.3-2.9 54 , 1.9-2.2 67 , 

1.7-1.9 12 , 

2.6-3.0 17 , 

3.0-3.1 54 , 

3.2-3.4 54 , 

5.2-6.0 9 , 

3.6-4.1 16 , 

T2.0-2.2 6 , T2.0-2.5 6 , 

Y3.1-3.8 11 , B5.3-5.7 13 , 

A2.3-2.4 21 , A1.9 21 , 

B2.8 23 , A2.2-2.5 38 , 

X2.8 63 Ash whole body, 10-12 

, 

dry† 

4 15 46 , 11-13 10 , 

11-14 15 , 11 37 X15-17 

, 

60 , B15-17 64 , 

X10-11 65 Ash vertebra, dry 43±2 

, 

2 , 27-30 4 42 7 , 48-53 8 , 

49 14 , 36-46 15 , 

B65 7 , P64 7 , S53 7 , I45 7 , 

A32 21 , F33-36 42 , 

Ash vertebra, 

fat-free, dry 

50-51 5 , 50-56 5 , 

50-51 54 , 53-58 58 , 

44-47 20 , 54 24 , 

52 25 , 50-55 26 , 

58-62 26 , 49-53 27 , 

53-60 44 , 53-57 50 56 

, 

37 , 50-52 54 , 65-66 16 , A48-52 3 , T39-42 6 , 

T45-46 6 , B56-58 18 , 

B40 23 , D52-54 41 , A54 56 , 

X46-52 58 , X47-50 62 , 

X52-56 63 , X37-38 65 , 

Ash scale & skin, 4.1-4.6 

wet 

39 , A7.6 21 , 

Ash scale, dry 16 7 , 20 37 , I43 7 , D46-51 41 , 

F41-44 42 , A46-48 56 , 

X27-29 65 , 

Ca whole body, 

wet 

0.52±0.12 1 , 

0.51±0.04 2 , 

0.48-0.52 17 , 

0.49-0.56 19 , 

0.72-0.76 39 , 

0.42-0.67 59 0.20-0.27 

, 

12 , 

0.73-0.80 17 1.6 

, 

9 , 

0.9-1.5 16 T0.25-0.48 

, 

6 , 

B0.93-1.13 13 , A0.35 21 , 

A0.45-0.49 38 , 

Ca whole body, 

dry† 

2.0-2.6 4 , 1.9-2.4 52 , 3.1-4.9 45 , 

4.2-4.4 46 , 

2.5-2.9 10 , 

2.3-3.4 15 , 

F2.6-3.5 40 , X4.6-5.8 60 , 

X3.7-3.8 65 , 

Ca vertebra, dry 17.9±1.8 2 , 9.0-11.4 4 20 7 , 18-21 8 , 

17 14 , 12-18 15 B31 

, 

7 , P29 7 , S25 7 , I22 7 , 

B22 13 , A12 21 , F11-15 42 , 

A12-21 55 Ca vertebra, 17-18 

, 

fat-free, dry 

54 , 9.8-9.9 58 , 14-15 20 , 20-21 26 , 

9-15 43 , 15-18 45 18 

, 

54 , 21-23 16 , A18-19 3 , T12-13 6 , 

T14-15 6 , B20-22 18 , 

B14 23 , F18-19 40 , 

D13-18 41 , A22 56 , 

I9.3-14.5 58 , 

X7.0-13.5 58 , X17 63 , 

X13-14 65 , 

Ca scale, wet 3.15±0.49 1 Ca 

, 

scale & skin, 1.4-1.7 

wet 

39 , A2.4 21 , 

Ca scale, dry 8.3 7 , I28 7 , F18 40 , D16-19 41 , 

F14-16 42 , A19 56 , 

X7.9-8.6 65 , 

Ca plasma, 

serum, wet* 110-12839 , 118±48 47 , 137-148 26 , 97-104 12 , 

95 14 , 

95-148 30 , 

80-160 48 A115 

, 

21 , B100 23 , 

F130-220 40 , F150 42 , 

X34-37 51 , A145 56 , 

81

P whole body, 

wet 

P whole body, 

dry† 

0.48±0.10 1 , 

0.47±0.03 2 , 0.37 17 , 

0.43-0.47 19 , 0.48 34 , 

0.43 36 , 0.52-0.54 39 , 

0.38-0.43 53 , 

0.41-0.59 59 , 

0.39-0.41 67 , 

1.9-2.3 4 , 1.6-2.0 52 , 2.1-2.8 45 , 

2.6-2.7 46 , 


0.16-0.19 12 , 

0.56-0.64 17 , 

1.6-1.9 10 , 

1.7-2.8 15 , 

1.8 37 , 

P vertebra, dry 8.8±0.8 2 , 5.6-6.5 4 , 7.6 7 , 

9.0-9.3 8 , 

9.5 14 , 

P vertebra, 

fat-free, dry 

9.0-9.7 54 , 3.7-4.9 58 , 

10.3-10.6 67 , 

8.5-8.9 20 , 

9.6-9.9 24 , 

8.5-9.2 25 , 

10.1-10.6 26 , 

9.0-9.7 26 , 

11.9-12.7 27 , 

1.8-6.5 43 , 

7.8-9.1 45 , 

9.8-10.5 50 , 

6.2-8.4 15 , 

9.4 37 , 

9.0-9.5 54 , 

0.92 9 , 

0.7-0.8 16 , 

11.5-12.3 

16 , 

T0.40-0.49 6 , 

B0.72-1.15 13 , A0.43 21 , 

A0.58 28 , A0.48-0.51 38 , 

X0.43-0.44 62 , 

F2.1-2.4 40 , X3.0-4.0 60 , 

B1.9-2.4 64 , X1.5-1.6 65 , 

B13.2 7 , P10.9 7 , S10.2 7 , 

I10.7 7 , B11-13 13 , 

A6.6 21 , F6.2-7.8 42 , 

A7-13 55 , 

A9.1-9.6 3 , T7.6-7.8 6 , 

T8.5-8.8 6 , B9.7-10.5 18 , 

B7.8 23 , F9.8-10.4 40 , 

D9.8-10.4 41 , A10.7 56 , 

I2.6-4.7 58 , X3.5-5.3 58 , 

X8.7-9.3 63 , X8.0-8.2 65 , 

P scale, wet 1.81±0.34 1 P 

, 

scale & skin, 0.87-1.02 

wet 

39 , A1.56 21 , 

P scale, dry 1.8 7 , 3.9 37 , I11.2 7 , F9.7-10.1 40 , 

D9.7-10.1 41 , F8.2-8.7 42 , 

A9.4-9.8 56 , X5.6-5.8 65 , 

Pi plasma, 

serum, wet* 

87±16 2 , 86-99 29 , 

123 31 , 153-171 35 , 

127±38 47 , 146 49 , 

200-230 61 , 121-140 66 , 

90-104 67 18-22 

, 

22 , 

165-184 26 , 

2.1-2.4 27 , 70 57 , 

69-77 12 , 57 14 , 

68-121 30 , 

70-120 48 , 

40-100 57 , 

A78-96 3 , B200 23 , 

Y58-137 33 , F230-247 40 , 

F96-130 42 , X260-320 51 , 

A63-76 56 , K100-240 57 , 

X109-117 62 , X47 63 , 

Ptotal plasma, 

serum, wet* 

202-216 29 , 167 31 , 

307-328 35 , 450-590 39 220 

, 

57 , 373-606 30 , 

240-510 57 , 

Y378-712 33 , A515 21 , 

K280-520 57 , 

Ptotal whole blood, 1315±83 

wet* 

1 , 1270 31 , 

790-1150 31 , 

770 57 , 800 32 , 

570-1200 57 , 

K520-1300 57 , 

Ca/P whole body 1.0-1.2 4 , 1.1-1.2 19 , 1.25-1.42 12 , 

1.1-1.5 15 , 

1.7 9 , 

0.5-0.8 16 T0.68-0.98 

, 

6 , 

B0.86-1.46 13 , 

A0.93-0.97 38 , 

Ca/P vertebra†† 1.6-1.8 4 , 1.8-1.9 54 , 2.0-2.2 8 , 

1.8 14 , 

1.9-2.2 15 , 

1.9-2.0 54 1.8-1.9 

, 

16 , T1.6-1.7 6 , T1.7 6 , 

B1.7-2.0 13 , B2.0-2.2 18 , 

B1.8 23 , D1.2-1.8 41 , 

A1.6-1.8 55 Ca/P plasma, 

serum 

1.7 

, 

14 , 

Codes for Other Species: A (Atlantic salmon); C (coho salmon); D (red drum); T (chum salmon), S (seabass); B 

(seabream); P (cod); I (sardine); Y (yellowtail); F (sunshine or striped bass); K (pike); X (none of these, see ref.). 

† Note that values expressed as whole body, dry basis, are inaccurat e since the body fat (and wat er) content is highly 

influenced by the dietary P level. 

†† Ca/P ratio in the bone (or bone ash) is invalid to diagnose P status of fish. 

* Values are express ed as mg/L. Levels are known to be highly variable depending on the dietary P level, hours 

after feeding, diet composition, and (especially for Pi) the analytical method. 

References and approx. fish sizes analyzed: 1 (Shearer 1984; 10-1822 g), 2 (Shearer & Hardy 1987; 180 g), 3 

82

(Vielma & Lall 1998a; 40 g), 4 (Ogino & Takeda 1978; 3.5 g); 5 (Ketola & Richmond; 110 and 28 g); 6 (Watanabe 

et al. 1980b; 5 and 9 g); 7 (Shimada & Kaneda 1937; size unknown); 8 (Takeuchi & Watanabe 1982; 13 g); 9 

(Satoh et al. 1984; 44 g); 10 (Sato et al. 1997; 9-40 g); 11 (Shimeno et al. 1994; 42-76 g), 12 (Shitanda & Ukita 

1979, Shitanda et al. 1979; 30-47 and 250 g); 13 (Satoh et al. 1998; 30 g), 14 (Yone & Toshima 1979; 26 g), 15 

(Ogino & Takeda 1976; 11 g), 16 (Watanabe et al. 1980a; 32 g), 17 (Ogino & Kamizono 1975; 5 g), 18 (Watanabe 

et al. 1984b; 600-1000 g), 19 (Lall & Bishop 1979; 100 g), 20 (Robinson et al. 1987; 11 g), 21 (Baeverfjord et al. 

1998; 5-60 g and 325 g), 22 (Eya & Lovell 1998; 24 g), 23 (Sakamoto & Yone 1973, 1978; 89 g), 24 (Li & 

Robinson 1997; 72 g), 25 (Lovell 1978; 5 g), 26 (Wilson et al. 1982; ca.45 g), 27 (Eya & Lovell 1997; ca.600 g), 

28 (Asgard & Shearer 1997; 3.5 g), 29 (Phillips et al. 1957; ca.300 g), 30 (Field et al. 1943; 1350 g), 31 (Phillips et 

al. 1961; yearling and 9 g), 32 (McCay 1930; size unknown), 33 (Shimizu et al. 1963; 151-1534 g), 34 (Podoliak 

& Smigielski 1971; 48 g), 35 (McCartney 1971b; fingerling), 36 (Phillips et al. 1964; 20 g), 37 (Schäfer et al. 

1995; 40 g?), 38 (Nordrum et al. 1997; 10 g), 39 (Skonberg et al. 1997; 5 g), 40 (Brown et al. 1993; ca.20 g), 41 

(Davis & Robinson 1987; 16 g), 42 (Dougall et al. 1996; 15-440 g), 43 (Launer et al. 1978; 6-90 g), 44 (Andrews 

et al. 1973; 180 g), 45 (Dove et al. 1976; fingerlings), 46 (Lovell & Li 1978; 3.5 g), 47 (Hille 1982; mean ± SD of 

13-26 independent studies), 48 (Nakamura 1982; 300 g), 49 (Rodehutscord 1996; 200 g), 50 (Li et al. 1996; 85 g), 

51 (Firdaus & Jafri 1996; 22 g), 52 (Hardy et al. 1983; 55 g), 53 (Wiesmann et al. 1988; 70-380 g), 54 (Ogino et al. 

1979; 9-15 g), 55 (Poston & Ketola 1989; maturing female), 56 (Vielma & Lall 1998b; 150 g), 57 (McCay 1931; 

size unknown), 58 (Hara 1930; 4 yr old Scomber sp., 2.5 yr old trout, no info. for sardine), 59 (Satoh et al. 1987; 2 

g

Table 4. Approaches to Reducing Phosphorus Pollution of Aquaculture 

Reducing P excretion by fish 

Reducing Fecal P excretion 

1. Increase fat in diet (increase energy density of diet) 

2. Use low-P (= low-ash or deboned) fish meal: Low-P fish meal is generally more costly than regular fish meal. 

3. Reduce fish meal / Increase plant protein sources: Reducing fish meal in diet generally reduces feed 

palatability, feed intake, and fish growth rate. 

4. Phytase supplementation: Phytase activity is low at “body temperature” of coldwat er fishes, making its 

supplementary effect relatively low. Phytase activity is low at GI pH of agastric fishes (e.g., carps). 

(a). microbial sources (heat-resistant phytases now available) 

(b). endogenous sources (e.g. wheat bran is high in phytase content) 

5. Use low-phytate mutant grains: Feasible if mutant grains are competitive in price and nutritional value. 

6. Dephytinization (by incubating grains with phytase, or washing grains with water preferably weakly acidi fied 

water, or allowing germination / acid ferm entation, etc.): Additional cost for the treatments. 

7. Dietary acidification: Few studies have been conducted in relation to dietary P-utilization. 

(a). of fish meal-based diets: Dietary P should not exceeds the requirement (the excess will be urinated). 

(b). of grain-based diets supplemented with phytase: Useful for agastric species and/or to save phytase. 

8. Finishing feeds (low-P): Effective during the final grow-out to pre-harvesting period when fish consume most 

feed in quantity during its life cycle. 

9. Increase intestinal P absorption by hormones (e.g., calcitriol): Approval needed for practical use; cost 

prohibitive; not yet studied in fish. 

10. Transgenic fish: Approval by consumers and environmentalists unlikely. 

(a). secrete phytase on their own (already made in rats and pigs) 

(b). overexpress critical genes in P digestion, absorption or metabolism--- e.g., NaPi cotransporters, 

1alpha-hydroxyl ase (CYP27B1), H + /K + ATPase (proton pump), etc.--- in GI tract, kidney, or other tissues. 

Reducing Urinary P excretion 

1. Reduce dietary available P level* to the minimum requirement of fish** 

*available P level depends on the forms of P in each ingredient, numerous interactions of P with other dietary 

components, total dietary P content, and the species. 

**P requirement depends on the fish growth rate; thus P-requirement in a diet is variable depending on the 

feed quality, feed intake, fish size, physiological state, cultural conditions, etc. 

2. Increase renal P reabsorption by hormones; see above 

Collecting fecal P in effluent water: Periodic cleaning, vacuuming, and disposal needed 

1. Use settling ponds, settling or collecting device 

2. Use fecal binders to stabilize feces (prevent disintegration) in pond (e.g. CMC, alginate, benthonite, clay, etc.) 

3. Select ingredients that can produce firm stable feces (wheat, wheat gluten, etc.) 

4. Avoid ingredients that produce diarrhea or amorphous feces (fish meal, soybean meal, corn gluten, etc.) 

5. Use recirculating aquaculture system: Expensive in water filtration, conditioning, and disease control. 

Collecting urinary or soluble P in effluent water: Expensive and ineffi cient 

1. Use P-binding chemicals, anion-exchangers, or other resins 

2. Hydroponics 

3. Polyculture with filter-feeders or mollusks in closed ponds 

4. Use recirculating aquaculture system: see above. 

Good Management Practices 

1. Minimize / Collect uneaten feeds, Remove fines (powder) 

2. Avoid net cage farming: Feed loss is typical; Local accumulation of feed and fecal matter affects ecosystems. 

3. Avoid automatic feeder: Feed loss is typical. 

4. Use floating feeds: Feed loss can be minimized. 

5. Monitor effluent P level. 

6. Monitor fish P status periodically based on body P indicators (see Table 2, 3). 

7. Education for GMP and responsible management practices: Feed fish, Do not feed ponds or cages. 


84

Figure 1. Ventral view of P-defi cient common carp sampled at a commercial fish farm: left and middle fish--- 

P-deficient; right fish--- P-adequate. Cranial malformation, especially the mandible, is characteristic in P 

deficiency in common carp. 

Figure 2. The same fish: Vertebral column. Note the absence of rhodosis or scoliosis. “Snaking ribs” is one 

of the characteristic signs of P deficiency. 

Figure 3. The same fish: The abdominal ribs. Deformity of the ribs is a typical sign of P-deficiency in fishes. 

Malformation of the bones does not mean that the fish is “currently” P-deficient. The bones might be deformed 

anytime in the past, especially during early stages of the development when dietary P requirement was high. 


85

Figure 4. Urinary P excretion in rainbow trout fed a high-P diet. Urine was collected continuously from fish 

confined in a metabolic chamber using a catheter. Fish was fed every 24 hours (vertical line). 

Amount of Urine (% of BW/h) 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

0 

8 

16 

Urinary P (Result, Expt.3) 

Amount of Urine 

P Concentration 

24 

32 

40 

48 

56 

64 


72 

80 

88 

96 

10 4 

112 

Hours in a Metabolism Chamber 

Figure 5. Urinary excretion of P as an indicator for estimating dietary P requirement of large rainbow trout. 

The urinary response to dietary P is very rapid, which is useful when working with large or adult fish. 

P Concentration (mg/L tank water) 

Requirement of P (Result, Expt.5) 

1.4 

Day 0 

1.2 

1.0 

0.8 

0.6 

1.4 Day 3 

1.2 

r=0.984 

1.0 

n=5 

0.8 

0.6 

Req. 0.698 

1.4 Day 6 

1.2 

r=0.987 

1.0 

n=5 

0.8 

0.6 

Req. 0.725 

0.4 

0.4 

0.4 

0.2 

0.2 

0.2 

0.0 

0.0 

0.0 

1.4 1.4 Day 12 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

Day 9 

r=0.984 

n=5 

Req. 0.627 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

r=0.988 

n=5 

Req. 0.695 

12 0 

128 

13 6 

144 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 

P Concentration in Urine (mg/L) 

86

Figure 6. Pyloric caeca are the major absorptive organ of dietary P in trout. Pyloric caeca repres ent 

approximately 70% of the total gut absorptive surface area in rainbow trout. Many species of fish have pyloric 

caeca. 


87

Review: Phosphorus in Fish Nutrition

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