Phosphorus bioavailability in diets for growing horses

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Livestock Science 116 (2008) 90 – 95 www.elsevier.com/locate/livsci

Phosphorus bioavailability in diets for growing horses A.A.M.A. Oliveira a,⁎, C.E. Furtado b , D.M.S.S. Vitti c , F.D. Resende d , S.L.S Cabral Filho e , H. Tosi f , B. Winkler g a

Universidade Estadual do Oeste do Paraná, Departamento de Zootecnia, UNIOESTE, Rua Pernambuco 1777, Marechal Candido Rondon, PR, 85960-000, Brazil b Universidade Estadual de Maringá, Departamento de Zootecnia, UEM, Av. Colombo,5790, Maringá, PR, 87020-900, Brazil c Animal Nutrition Laboratory, Centro de Energia Nuclear na Agricultura, Caixa Postal 96, CEP 13400-970, Piracicaba, SP, Brazil d Apta Regional Pólo Alta Mogiana, Cx Postal 35, Colina, SP, 14770-000, Brazil e Universidade de Brasilia-UNB-Faculdade de Agronomia e Veterinaria, campus Darcy Ribeiro, Asa Norte, CEP: 70910-900, Brazil f Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Zootecnia de Ruminantes, UNESP, Rodovia Carlos Tonanni Km 5, Jaboticabal, SP, 14870-000, Brazil g University of Plymouth, Department of Agriculture and food studies, Seale-Hayne Faculty, Newton Abbot, TQ12 6nQ, England, United Kingdom Received 24 February 2006; received in revised form 2 August 2007; accepted 3 September 2007

Abstract A study of phosphorus (P) metabolism was carried out using 12 month old Brasileiro de Hipismo breed of horses to determine the P bioavailability available from feeds commonly fed to horses in Brazil. Five different diets were formulated to contain approximately equivalent levels of crude protein and digestible energy, as well as to supply at least 22 g P/horse/day (NRC, 1989). All 5 diets contained 40% Bermuda coastal hay plus 60% concentrate. The 5 different concentrates contained: C1 (corn + cottonseed meal) C2 (corn grain + soybean meal) C3 (corn + sugarcane yeast), C4 (oat + cottonseed meal), and C5 (oat + soybean meal). The radioisotope 32 P was injected with 30 MB. Blood, feces and urine were collected for 7 days to evaluate endogenous fecal P and true absorption. Analysis of variance of P intake showed differences due to dietary effects (P b 0.05). Concentrate C3 had the lowest intake (79.68 mg/ kg BW). All of the diets produced positive P retention. Absolute values for P concentrations in plasma, urine, feces and endogenous feces did not vary between diets. Values for endogenous fecal P were independent of the level of P intake, so the correlation between P intake and P endogenous was not significant. P bioavailability values were 50.75; 40.98; 43.50; 51.03 and 57.68% for diets C1 through C5, respectively. However, differences in P bioavailability were found (P b 0.05) between diets. Diets C2 and C3 had lower P bioavailability than the other diets. The P bioavailability of all dietary treatments in this study exceeded NRC (1989) postulations of 35% true P absorption in diets not supplemented with inorganic P. The results of this study indicate that inorganic P supplementation is not needed for growing yearlings fed common Brazilian feeds. Considering the high cost of P supplementation and the risk of environmental P contamination, inorganic phosphorus supplementation for growing yearlings may not be required. © 2007 Elsevier B.V. All rights reserved. Keywords: Phosphorus; Bioavailability; Equine; True absorption

⁎ Corresponding author. E-mail addresses: [email protected] (A.A.M.A. Oliveira), [email protected] (C.E. Furtado), [email protected] (D.M.S.S. Vitti), [email protected] (F.D. Resende), [email protected] (S.L.S.C. Filho), [email protected] (B. Winkler). 1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.09.010

1. Introduction The adequate growth of the skeleton is vitally important in the development of young horses. Any aberration in the development of the skeleton can

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seriously impair the horse's ability to perform to maximum genetic potential. For some time, researchers have given attention to nutrition as a causative factor in DOD (Developmental Orthopedic Disease) (Teeter et al., 1967, Cunha, 1991). Although several nutritional factors have been implicated as causes, phosphorus (P) adequacy has been shown to be critical for the development and integrity of the skeleton in growing horses (Hintz and Schryver, 1976). In spite of its importance in development, other factors need to be considered when trying to achieve the correct levels of phosphorus in food sources. Studies have concluded that exercise of the horse also influences the mineralization of bones and bone quality. Nielsen et al. (1998) reported that exercise can influence P turnover and bone remodeling in horses starting in training. Also, phosphorus supplementation is not only costly, (Furtado, 1996) but can also cause undesirable imbalances among minerals when over supplemented (Honoré and Uhlinger, 1991). In addition, feeding reduced levels of P, to the extent possible, diminishes the environmental impact of equine operations and the land area required for manure disposal. Common horse feedstuffs that are relatively high in P include cereal grains, some of the oilseed meals (cottonseed meal and soybean meal) and some alternative feeds (sugarcane yeast among others). However, most of the organic phosphate in feeds is in the form of P phytate, which is relatively, although variably, available for absorption in non-ruminants. Several studies have investigated the digestion and metabolism of P in horses (Schryver et al., 1971, Kichura et al., 1983), and results have shown that P bioavailability ranged from 30 to 45%. They also reported that P bioavailability can be influenced by P intake, age of the animals and calcium: phosphorus ratio. Hintz (1983) reported some values of P bioavailability in a few feeds like corn grain (32.0%) and oat grain (40.0%). So far, no additional studies have been conducted regarding this issue. P bioavailability can be determined by phosphorus radioisotope studies (Georgievskii, 1982). In Brazil, the first P radioisotope studies on horses were conducted by Furtado (1996) using 32P as the phosphorus isotope. The author determined P availability in growing horses supplemented with inorganic P sources from rock phosphates, dicalcium phosphate and bone meal. He found that in spite of different inorganic sources of P, bioavailability was the same. The results ranged from 29.0 to 34.0% when the P intake ranged from 89.35 to 105.31 mg of P/ kg of BW. The large intestine of the equine is considered to be the primary site of P absorption. Large intestine microbes are

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capable of producing phytase, an enzyme capable of releasing P from its bond with phytate (organic source). As the horse itself does not produce phytase, microbial digestion is the primary mode by which P is available for absorption (Schryver et al., 1972). In rats, diets high in phytate P appear to produce adaptations in the microbes over time to improve P degradation (Fairweather-Tait, 1997). Matsui (1999) reported that phytate binding of P does not hamper P absorption in equine. The more microbes degrade phytate, the more phosphorus will be free to be absorbed in the large intestine. With this information Matsui (1999) confirmed that the P in the phytate form does not present problems of absorption for horses, since it is absorbed in ratio to its degradation predominant in the large intestine. The objective of this study was to determine the P bioavailability (true absorption) available from feeds that are not supplemented with any additional inorganic P source and are commonly fed to horses in Brazil. 2. Materials and methods 2.1. Experimental procedure This study was conducted at the Animal Nutrition Laboratory at the Center for Nuclear Energy in Agriculture (CENA), University of Sao Paulo. Eight male horses were used (Brasileiro de Hipismo breed) in this research. The average age of the horses at the beginning of the experiment was 10 months and the mean live weight was 220 kg. The animals were fed five diet treatments which were formulated to contain approximately equivalent levels of crude protein and digestible energy, as well as to supply at least 22 g P/horse/day according to NRC (1989). All five diets contained 40% Bermuda Coastal hay plus 60% concentrate. The concentrate portions of the five diets were composed of 15% wheat bran plus 4% of a mineral mix formulated to balance the naturally occurring P of the diets to a Ca: P ratio of 1.25:1. The mineral mix also met trace mineral requirements. The remainder of the concentrate, was comprised of different cereal grains and protein supplements combined to provide approximately 2.5 M cal DE/kg and approximately 20% CP. The concentrates contained the following ingredients: C1 (corn grain + cottonseed meal), C2 (corn grain + soybean meal), C3 (corn grain + sugarcane yeast), C4 (oat grain + cottonseed meal), and C5 (oat grain + soybean meal). The five diets (see Table 1) were fed at the rate of 2.5% of body weight and all diets were formulated to meet 100% of the requirements for a yearling (NRC, 1989). Because of the limited number of animals (8), limited facilities to conduct the radioisotope trial with all animals, and the necessary time break intervals between treatments; this experiment was distributed into three periods. Each experimental period was divided into a phase of 15 days for diet adaptation and 7 days for the trial of metabolism of phosphorus 32P radioactive. There was a 30 day break between the experimental periods. The

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Table 1 Feed ingredients and composition of the experimental diets fed to yearling horses a Item

Diets

Ingredients (%)

Diet 1

Diet 2

Diet 3

Diet 4

Diet 5

Bermuda Coastal hay Corn Grain Oat Grain Wheat bran Soybean meal Cottonseed meal Sugarcane yeast Mineral mixture b Total

40.0 30.0 – 9.0 – 18.6 – 2.4 100.0

40.0 34.2 – 9.0 14.4 – – 2.4 100.0

40.0 29.4 – 9.0 – – 19.2 2.4 100.0

40.0

40.0

32.4 9.0 – 16.2 – 2.4 100.0

36.0 9.0 12.6 – – 2.4 100.0

Chemical composition

C1

C2

C3

C4

C5

Dry Matter Organic Matter c Ash c Ether extract c Crude fiber c NDF c ADF c Crude protein c DE (M cal/kg DM Ca c Pc

90.80 92.23 7.77 4.14 6.93 44.95 10.33 22.40 2.43 1.05 0.87

89.93 93.42 6.58 4.27 4.88 47.17 6.60 21.49 2.51 0.95 0.75

90.46 93.80 6.20 3.46 4.07 39.11 4.43 20.38 2.49 1.00 0.72

91.06 93.77 6.23 4.72 13.20 46.90 17.80 20.00 2.41 1.00 0.82

90.61 94.12 5.88 4.17 10.73 39.42 14.33 23.84 2.46 1.03 0.75

Concentrates

a

Bermuda Coastal hay Chemical Composition (% Dry matter basis): DM 89.61;OM 93.16; Ash 6.84; EE 2.35; CF 31.16; NDF 78.45; ADF 38.92; CP 9.06; DE 1.87 M ca/kg; Ca 0.40; P 0.11. b The mineral mixture composition per kg of product: 1.500 mg of Zn; 250 mg of Cu; 1000 mg of Fe; 12.4 mg of Co; 20 mg of I; 2.25 mg of Se and 0.72 g of F. c Percentage of DM.

diets offered were divided in advance into three daily meals of equal size to be given at 8:30 h; 12:30 h and 16:30 h. Also, a metabolism crate, was specially designed for study using radioisotopes. On the first day of metabolic study each animal was given, as a single dose via the right jugular vein, 30 M Bq of 32 P in 1 ml of sterile isotonic saline (8.5 g/L NaCl). After isotope

administration, blood samples, were drawn, from the left jugular vein into vacutainer tubes (10 mL heparin glass tube) containing sodium heparin at 24 h intervals for 7 days. Once, most of the mobile P in blood can be found in plasma, the blood was centrifuged (300 rpm during 10 min) and plasma was removed for P analysis. Nine milliliters of trichloroacetic acid (100 g/L) was added to 1 mL of plasma for protein precipitation. After 10 min the material was filtrated and inorganic P was determined by colorimetric analyses (Fiske and Subbarrow, 1925). (Table 2). During the 32P metabolism trial, a total collection of feces and urine was made each morning for 7 days. after isotope administration. An aliquot of 5% of total feces and urine outputs were sampled and stored for further analysis. Feces samples (1 g) were dried overnight (105 °C) and ashed (500 °C for 8 h). The ash was dissolved in concentrated HCl, and P content was determined by the colorimetric method (Sarruge and Haag, 1974). An analogous procedure was used to determine P content of food intake. Urine samples (30 mL) were acidified using 100 mL of HCl (12 N). They were then dried (55 °C) and made into ashes (500 °C). Samples of the ash were diluted (3 N HCl) and volume was made up to 10 mL (Morse et al., 1992). Inorganic P was determined using vanadate-molybdate reagents (Sarruge and Haag, 1974). For radioactivity measurements, 1-mL plasma and urine samples were added to 19 mL of distilled water in counting vials. For feces, ashed fecal samples (1 g) were dissolved in 18 N H2SO4 and placed in counting vials. The volume was up to 20 mL. Radioactivity of 32P was measured in a Packard Liquid Scintilation Spectrometer (Model 2450B, A. Canberra Company) using Cerenkov radiation. Specific activities in plasma feces and urine were determined according to Lofgreen and Kleiber (1953), which is the percentage of injected activity per milligram of P. 2.2. Statistical methods A mathematical model was adopted in this study for the eight animals. A applied orthogonal contrast was used on the 5 tested diets where Xi (I = 1,4) and Pj (j = 1,3) are the applied orthogonal contrasts on the three used periods. A mathematical model was used for the statistical analysis (y = b0 + Σ bi Xi + Σ bj Pj) where y is the estimate of the response measured

Table 2 Mean of values of parameters from phosphorus metabolism Parameter\treatment

C 1 corn/cott.

C2 corn/soy

C 3 corn/yeast

C4 oat/cott

C 5 oat/soy

P intake (mg/kg BW) P plasma (mg/100 ml) P urine (mg/kg BW) P feces (mg/kg BW) P endogenous (mg/kg BW) P absorbed (mg/kg BW) P bioavailability P retained (mg/kg BW) Body weight (kg)

113.08a 5.83a 1.93a 63.78a 8.00a 57.30a 50.75a 47.43a 255.75a

95.98b 5.75a 6.40a 64.38a 7.78a 39.38bc 40.98b 25.20b 261.80a

79.68c 5.90a 1.38a 51.23a 6.73a 35.17c 43.50b 27.05b 288.93a

105.20a 5.70a 4.00a 61.55a 9.80a 53.43ab 51.03a 39.95b 258.93a

99.43b 7.10a 6.85a 51.30a 9.78a 57.95a 57.68a 41.28b 278.00a

abc

= Values followed by identical letters on the same line are not significantly different (P N 0.05).

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considering diet i and period j; b0 is the linear coefficient of the model;and bi and bj are the coefficients of the independent variable Xi (diets) and Pj (Periods).

3. Results and discussion Table 2 shows mean values results of parameters measured for phosphorus metabolism. 3.1. Intake Analysis of variance of P intake showed differences due to dietary effects (P b 0.05). The animals that received C3 had the lowest P intake (79.68 mg/ kg BW or 23 g P/head/day) of all of the concentrates. The lowest intake value of P was equal to the lowest intake reported by Furtado (1996) when Tapira rock phosphate was supplemented (89.35 mg/kg BW). The animals that received C1 and C4 had the highest P intakes (113.08 and 105.20 mg P/kg BW or 29 g and 27 g of P/head/day, respectively). The highest P intake can be explained by the use of cottonseed meal as a protein source in both concentrates. It is rich in P (1.08% of total P) while soybean meal has only 0.60% of total P. Even without the use of inorganic phosphate sources in the present experiment, the P intake in all dietary treatments were considered adequate according to NRC (1989) for yearlings experiencing rapid growth (600 kg mature weight). It should be consumed at least 22 g of P head/day. The lowest intake presented in this study was shown by C3. A colt with an average body weight of 289 kg consumed a total of 23 g of P/ head/day. 3.2. In the plasma It did not present a significant difference (P N 0.05) between concentrates, despite the fact that C5 had showed the highest value of P in the plasma (7.10 mg/ 100 ml). All the concentrates in the study maintained adequate levels (above of 3.22 mg/100 ml) of phosphorus in the blood. According to McDowell (1992), the P plasma concentration values can range from 4 to 9 mg/ 100 ml. Kichura et al. (1983) had reported similar values to those found in C5. When the diets had a rich P intake of 115 to 128 mg/kg BW/day they reported values of 8.3 and 7.5 mg of P/100 ml of plasma, respectively. 3.3. Excreted in urine There was no significant difference in P excreted in urine (P N 0.05) among the concentrates. Since the P

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intake obtained in this study was considered adequate, the results of P excreted in urine have been inferior. Schryver et al. (1971) has indicated that horses fed adequate P diets (0.2% P) excreted 1–2 mg P/kg/1.The authors reported values of P excreted in urine of 19 mg/kg BW, when the daily P intake was 108 mg/kg BW. This is equivalent to 17.6% of the total P intake. In this study the average amount of P excreted in urine was equivalent to 4.1% of the total P intake. One of the factors that can influence P excreted in urine is the level of calcium in the diets, which is subordinate to the hormonal control, PTH. The calcium to phosphorus ratio adopted in this study remained constant and was 1.25:1 mean value in all of the diets. 3.4. Excreted in feces There was no significant difference in P excreted in feces between concentrates (P N 0.05). Even so the values obtained from C3 and C5, showed lower numerical values than the other concentrates. In contrast, Furtado (1996) showed high values of P excreted in the feces, when both testing under the same conditions as this study using inorganic phosphate sources, 76.57; 72.14, 82.96 and 81.02 mg/kg of BW, respectively, for Tapira rock phosphate, Patos of Minas rock phosphate, bicalcium phosphate and bone meal. The total P excreted in feces from this study represents about 59.57% of P intake which is lower than the value reported by Furtado (1996) at 79.8% of P intake. In contrast, the result of P excreted in feces obtained from this study can support the thesis of the main route of P losses in equine is through the feces. This agrees with Hintz (1983); Schryver et al. (1971); Hintz and Schryver (1973); Cymbaluck and Christensen (1986), who have concluded that the feces is a main route of excretion, especially when the animal does not consume diets with excessive levels of P. 3.5. Endogenous fecal losses of P There were no significant difference in endogenous fecal losses of P between concentrates (P N 0.05), although the animals that had received C3, have shown lower numerical values for this parameter. The mean values of endogenous fecal P obtained in this study for all the diets was about 8.42 mg P/kg BW and followed the variation of 6.0 to 13.0 mg/kg BW related by Schryver et al. (1971). Values for endogenous fecal P were independent of the level of P intake since the correlation between P intake and endogenous fecal P-values was not significant.

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3.6. Absorption The statistics analysis indicated significant difference (P b 0.05) and the animals that consumed concentrates C2 and C3 exhibited lower values of P absorption. The lower numerical value obtained C3 was possibly related to both the use of the sugarcane yeast and the lower P intake. Despite the highest P intake in C2, its low absorption can be related to the use of corn grain as the energy source, since when oat grain was combined with soybean meal the mean values obtained for P absorption had been statistically higher. Unfortunately, it was not possible to test the combination of oats with the sugarcane yeast to confirm the differences obtained in the P absorption. The mean values of phosphate absorption found in this study (48.65 mg/kg BW) were superior to the values reported by Hintz et al. (1973) where the phosphate absorption contained in wheat bran was determined to be about 38.08 mg/kg BW when not supplemented with inorganic sources. The correlation made between the P absorbed and the P intake was high and significant (r = 0.69) (P b 0.01), indicating that the total P present in feeds, including the phytate phosphorus had been totally absorbed. Our results partially agree with the observations of Van Doorn et al. (2004) who studied apparent P digestibility with horses and concluded that phytate phosphorus in feedstuffs is partially available. 3.7. Retained Analysis of variance of P retained showed differences due to dietary effects (P b 0.05). The animals that consumed C1, presented the highest retention of P, 47.43 mg/kg BW, (P b 0.05), when compared to the other concentrates. The biggest retention in C1 can be explained by the largest phosphorus intake (113.08 mg/ kg BW). The correlation between P retained and the P intake was positive and highly significant (P b 0.01) (r = 0.68). This study shows that the mean values of P retained in all the diets support a positive P balance. This fact reinforces the hypothesis that diets without supplementation with P inorganic sources do not always lead to a P deficiency once the P intake is adequate. Mean values obtained in this study were higher than the values reported by Furtado (1996), even when bicalcium phosphate (22.0 mg/kg BW) was used. 3.8. Bioavailability Differences in bioavailability were found (P b 0.05) between concentrates. P bioavailability (true absorp-

tion) values were 50.75; 40.98; 43.50; 51.03 and 57.68% for C1 through C5, respectively. Concentrates C2 and C3 had lower P bioavailability than the other concentrates. C5, had the highest numeric value of P bioavailability but was not significantly higher than C1 or C4. The use of sugarcane yeast and soybean meal as a protein source, combined with corn grain, was closest to the P bioavailability value recommended by NRC (1989). The use of cottonseed meal was an advantage because highest values of P bioavailability were obtained with C1 and C4 (50.75 and 57.68%, respectively). The mean P bioavailability values obtained in this study were higher than expected. According to Hintz (1983) and NRC (1989), P bioavailability values for horses are about 30 to 55%. If inorganic mineral sources are being used the values can be increased. Hintz and Schryver (1972) worked with mature ponies and basal diets supplemented with different inorganic P sources: bicalcium phosphate, bone meal, and limestone plus monosodium phosphate, they reported values of P bioavailability around 39.3 to 47.1%. Schryver et al. (1971), working with radioisotope 32P and equine reported 45% of P bioavailability using a basal diet with 0.7% of P supplemented with monosodium phosphate. This study showed higher values of P bioavailability with concentrates C1, C4 and C5: 50.75; 51.03 and 57.95%, respectively. The difference between the values found in this study and the literature may be explained by the methodology adopted. The lower P bioavailability value reported was obtained in a situation where the feed was fed separately and not combined with other as an ingredient in a balanced diet for equine. The other explanation is that most of the research using inorganic sources studied supplementation of basal diets in P instead of using regular or balanced diets that followed the nutritional requirements. Additional studies are necessary to confirm a metabolic adaptation inside the large intestine of horses that shows the ability to improve the P absorption. Fairweather-Tait (1997) have suggested this same adaptation in rats. The highest values of P bioavailability found in this study can be a confirmation of the work of Matsui (1999) which reported that total P inside the digestive system of equine, including phytate, was not a problem for P absorption. Moreover, van Doorn et al. (2004) has studied the effect of phytase supplementation on the availability of phytate bound P and concluded that phytase supplementation with feedstuffs did not present an influence on apparent phosphorus digestibility. This might imply a possible metabolic adaptation inside the digestive system of the horse.

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4. Implications The results of this study indicate that inorganic P supplementation is not needed for growing yearlings fed with common Brazilian feeds. Considering the high cost of P supplementation and the risk of environmental P contamination, results of this study indicate that inorganic phosphorus supplementation for growing yearlings may not be required. However, the diet has to be certified. Acknowledgements The authors wish to acknowledge Dr Flavio Dutra Resende and the Regional/Polo Alta Mogiana for the use of their animals, staff and facilities; the statistical advice provided by Dr. Ivan Sampaio; the staff of the Animal Nutrition Laboratory, Centro de Energia Nuclear na Agricultura, CENA/USP; and the methodology advice provided by Dr. Dorinha M.S.S Vitti. This study was of the result of a graduate research internship granted by the Brazilian National Research Council (CNPq). References Cunha, T.J., 1991. Horse. feeding and nutrition. Academic Press, San Diego. Cymbaluck, N.F., Christensen, D.A., 1986. Nutrient utilization of pelleted and unpelleted forages by ponies. Canadian Journal of Animal Science 66, 237–244. Fairweather-Tait, S.J., 1997. From absortion and excretion of minerals to the importance of bioavailability and adaptation. British Journal of Nutrition 78 (Suppl.2), S95–S100. Fiske, C.H., Subbarrow, Y., 1925. The colorimetric determination of phosphorus. Journal of Biological Chemistry 66, 375–400. Furtado, C.E., 1996. Avaliation of bioavailability and endogenous losses fecal for growing horses. Effects of inorganic sources and levels of phosphorus. Doctorial Thesis, Jaboticabal, Brazil. Georgievskii, V.I., 1982. The physiological role of macroelements. In: Georgievskii, V.I., Annenkov, B.N., Samokhin, V.T. (Eds.), Mineral nutrition of animals. Butterworts, London, pp. 91–170. Hintz, H.F., 1983. Horse nutrition. Arco Publishing, New York. Hintz, H.F., Schryver, H.F., 1972. Availability to ponies of calcium and phosphorus from various supplements. Journal of Animal. Science 34, 979–980.

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