Physiological Responses to Divergent Selection for Phytate Phosphorus Bioavailability in a Randombred Chicken Population1

Genetics Physiological Responses to Divergent Selection for Phytate Phosphorus Bioavailability in a Randombred Chicken Population1 P. K. Sethi,* J. P. McMurtry,† G. M. Pesti,* H. M. Edwards Jr.,* and S. E. Aggrey*2 *Department of Poultry Science, University of Georgia, Athens 30602-2772; and †USDA Animal Biosciences and Biotechnology Laboratory, Beltsville, MD 20705-2350 line, suggesting that the line differences may be the result of factors other than PBA. Glucagon and IGF-I have different relationships with feed conversion ratio in the high PBA line compared with the low PBA line. There was a significant correlation between PBA and T3 in the low line and between PBA and T4 in the high PBA line. Thyroid hormone levels may be an indirect indicator of PBA in growing chickens. The genes in the thyroid hormone pathway may be key in the identification of genes associated with PBA.

Key words: divergent selection, phytate phosphorus bioavailability, thyroid hormone, insulin-like growth factor, glucagon 2008 Poultry Science 87:2512–2516 doi:10.3382/ps.2008-00190

INTRODUCTION Phosphorus is the second most abundant mineral in the body with 80% of the total quantity found in the skeletal system. Deficiency of P can cause rickets, retarded growth, and other skeletal deformities. In poultry, P is an essential element for growth and development and plays an important role in metabolism of carbohydrates, lipids, and amino acids (Zhang et al., 2003). Therefore, it is important that birds should receive adequate amounts of available P in their diet to meet their metabolic demands. Poultry diets comprise plant-source ingredients such as cereal grains, cereal byproducts, and oilseed meals, and 60 to 80% of total P of plant origin exists as phytate P, where P is bound to phytic acid (Ravindran et al., 1994). Phytic acid (known as inositol hexaphosphate) reduces the bioavailability of P in monogastrics by forming insoluble (phytate) salts with divalent cations (Ca, Mg, Fe, Zn, and Mn) under weak acid to neutral conditions (Kratzer et al., 1959). Due to insufficient endogenous phytases (enzyme required to release the phosphate group from phytate to ©2008 Poultry Science Association Inc. Received May 11, 2008. Accepted June 25, 2008. 1 Supported by funds from US Poultry and Egg Association and State and Hatch funds allocated to the Georgia Agricultural Experimental Stations of the University of Georgia. 2 Corresponding author: [email protected]

make P available) in the digestive tract, poultry cannot fully utilize phytate P (Heuser et al., 1943), and as a result, lose P through their excreta. Exogenous phytases (from supplementation) interact with vitamin D, Ca, and other nutrients to improve phytate P utilization (Simons et al., 1990; Edwards, 1993; Ravindran et al., 1995; Kies et al., 2001). Several physiological parameters control P metabolism. Phosphorus, Ca, and vitamin D influence the absorption and metabolism of each other. The active form of vitamin D, 1,25 dihydroxycholecalciferol [1,25-(OH)2D3], stimulates Ca uptake. Any increase in Ca absorption results in an increase in Ca retention, which leads to greater P retention (Helander et al., 1996). Normal blood levels of Ca and P are under the hormonal control of the parathyroid hormone, vitamin D, and calcitonin. Laroche and Boyer (2005) suggested that hormones such as insulin-like growth factor (IGF)-I, thyroid hormones, and insulin increase tubular P resorption by the kidney. Quigley and Baum (1991) demonstrated that IGF-I microperfusion stimulates the absorption of phosphate in the rabbit proximal convoluted tubules. However, the effects of thyroid hormones on P resorption and serum P levels have been conflicting (Logan et al., 1941; Beisel et al., 1958; Kobe et al., 1999). A short-term (3-generation) divergent selection for phytate P bioavailability (PBA) in a randombred chicken population showed a modest response (Zhang

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ABSTRACT An investigation was conducted to study insulin-like growth factor (IGF)-I, IGF-II, insulin, glucagon, leptin, triiodothyronine (T3), and thyroxine (T4) levels in a chicken population divergently selected for P bioavailability (PBA). There were differences in growth and feed efficiency between the 2 lines. Concentrations of IGF-I, IGF-II, and T3 were significantly greater in the high PBA line compared with the low PBA line, whereas the reverse was true for glucagon. There were no correlations between IGF-I and II and PBA in either

DIVERGENT SELECTION FOR PHYTATE PHOSPHORUS BIOAVAILABILITY

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Table 1. Means (±SE) of BW, BW gain (BWG), feed consumption (FC), feed conversion ratio (FCR), insulin-like growth factor (IGF)-I, IGF-II, leptin, triiodothyronine (T3), thyroxine (T4), insulin, and glucagon concentrations in a chicken population divergently selected for high and low phytate P bioavailability (PBA) Trait

High PBA line
(n = 357)

PBA (%) BW (g) BWG (g) FC (g) FCR (g/g) IGF-I (ng/mL) IGF-II (ng/mL) Leptin (ng/mL) T3 (ng/mL) T4 (ng/mL) Insulin (pg/mL) Glucagon (pg/mL)

31.19 295.09 49.38 120.23 2.51 43.06 40.57 2.93 3.01 11.25 1,265.60 184.65

± ± ± ± ± ± ± ± ± ± ± ±

0.48 3.07 0.65 1.12 0.02 0.57 1.02 0.10 0.05 0.18 31.60 7.50

MATERIALS AND METHODS Birds and Selection A divergent selection was undertaken in the unselected random mating Athens-Canadian randombred (ACRB) chicken population (Hess, 1962). The growth, FC, and FCR of the base population have been reported previously (Zhang et al., 2003). Individual 4-wk BW, BWG, FC, and FCR during a consecutive 3-d period were measured. The experimental design, measurement of PBA, and results of the divergent selection and correlated responses have been described by Zhang et al. (2003, 2005a,b).

28.07 319.32 52.47 123.36 2.40 39.26 44.23 2.86 2.80 10.90 1,215.33 205.30

± ± ± ± ± ± ± ± ± ± ± ±

0.51 3.22 0.68 1.17 0.02 0.60 1.07 0.11 0.05 0.19 33.12 7.72

Pr > F 0.000 0.000 0.001 0.054 0.005 0.000 0.013 0.691 0.002 0.189 0.273 0.056

intraassay CV of 3.9% (Evock-Clover et al., 2002). The T3 and T4 were determined with intraassay CV of 2.5 and 2.8%, respectively (McMurtry et al., 1988). An aliquot of plasma was stored in the presence of 1,000 kIU of aprotinin for glucagon level determinations. Plasma glucagon was determined using commercial kits (Linco Research Inc., St. Charles, MO) with an intraassay CV of 1.9% (Richards and McMurtry, 2008).

Statistical Analysis The PROC GLM (SAS Institute, 1998) was used to test the effect of selected line after correction for sex and hatch effects, as there was no sexual dimorphism for PBA. The PROC CORR (SAS Institute, 1998) was used to test for correlation among hormones, BW, BWG, FC, and FCR within each selected line. The PROC MAX R2 procedure (SAS Institute, 1998) was used to determine hormones that significantly contributed to PBA, BW, BWG, FC, and FCR within each line.

RESULTS AND DISCUSSION Hormone Analysis At generation 3 of selection for PBA, 4 mL of blood was collected from each bird after the last day of excreta collection (wk 5) using EDTA as an anticoagulant. Blood was collected from 166 males and 191 females from 3 hatches in the high line and 167 males and 166 females from 3 hatches in the low line. The blood samples were centrifuged for 10 min at 503 × g, and the plasma was separated and stored frozen at −20°C. The frozen plasma was analyzed in duplicate for IGF-I, IGF-II, insulin, glucagon, leptin, triiodothyronine (T3), and thyroxine (T4) levels using homologous hormone assays. To avoid interassay variation, all samples were analyzed within one assay. Double-antibody radioimmunoassays were used to determine plasma concentrations of IGF-I with an intraassay CV of 2.8% (McMurtry et al., 1994), chicken IGF-II with an intraassay CV of 3.7% (McMurtry et al., 1998), insulin with an intraassay CV of 2.2% (McMurtry et al., 1983), and leptin with an

The physiological differences between the high and low PBA lines are shown in Table 1. There were significant differences in plasma IGF-I, IGF-II, glucagon, and T3 levels among the divergently selected lines. However, leptin, T4, and insulin levels were similar in both lines. The hormonal differences in the divergent lines can also be explained by differences in growth and feedrelated traits as BW, BWG, FC, and FCR, which are all significantly different among the low and high PBA lines. The correlation coefficient among PBA, growth and feed traits, and hormonal levels for each divergent line is given in Table 2. Even though there is no experimental evidence to suggest that the high PBA line is the result of increased endogenous phytase, the high phytate P retention could be due to increased resorption of P. However, there is evidence to suggest that IGF-I significantly increases P resorption in the renal tubules (Caverzasio et al., 1990; Hirschberg et al., 1995; Laroche and Boyer, 2005). Other researchers have dem-

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et al., 2005a), and the cumulative divergent correlated responses for BW, BW gain (BWG), feed consumption (FC), and feed conversion ratio (FCR) to divergent selection for PBA have been reported (Zhang et al., 2005b). In the current study, the hormone dynamics in the divergently selected lines for PBA were investigated.

Low PBA line
(n = 333)

0.97 <0.00 −0.40 <0.00 −0.50 <0.00 0.16 0.01 0.02 0.79 0.12 0.05 0.06 0.36 0.04 0.50 0.01 0.09 0.07 0.24

0.88 <0.00

BWG

−0.21 0.00 −0.58 <0.00 0.20 0.00 0.03 0.63 0.10 0.10 0.02 0.76 0.04 0.47 0.11 0.07 0.07 0.25

0.93 <0.00 0.97 <0.00

FC

−0.35 <0.00 0.11 0.07 0.05 0.41 −0.17 0.01 −0.16 0.01 0.01 0.87 0.04 0.50 −0.02 0.77

−0.44 <0.00 −0.72 <0.00 −0.52 <0.00

FCR

−0.12 0.05 −0.04 0.49 0.03 0.65 0.07 0.31 0.04 0.50 −0.21 0.00 −0.02 0.78

−0.50 <0.00 −0.26 <0.00 −0.44 <0.00 −0.37 <0.00

PBA

For each pair, the top number is the correlation coefficient and bottom number is the P-value.

1

T4

0.90 <0.00 0.93 <0.00 −0.18 0.00 −0.58 <0.00 0.13 0.03 −0.00 0.99 0.12 0.05 −0.02 0.74 0.01 0.89 0.13 0.04 −0.01 0.86

BW

−0.20 0.00 0.04 0.56 −0.30 <0.00 0.20 0.00 −0.24 <0.00 −0.14 0.02

0.16 0.01 0.23 0.00 0.20 0.00 −0.17 0.00 −0.02 0.72

IGF-I

−0.08 0.20 0.01 0.91 −0.05 0.45 0.27 <0.00 0.41 <0.00

0.07 0.21 0.08 0.17 0.07 0.24 −0.08 0.20 −0.02 0.74 −0.16 0.01

IGF-II

−0.18 0.00 −0.23 0.00 0.08 0.21 −0.23 0.00

0.09 0.11 0.05 0.41 0.07 0.22 0.01 0.83 −0.07 0.27 −0.03 0.57 −0.07 0.24

Insulin

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T3

Leptin

Glucagon

Insulin

IGF-II

IGF-I

PBA

FCR

FC

BWG

BW

Trait

−0.20 0.00 0.19 0.00

−0.05 0.39 −0.11 0.07 −0.09 0.13 0.11 0.07 −0.04 0.50 0.06 0.34 0.15 0.01 −0.22 0.00 −0.05 0.39

0.13 0.04 0.17 0.01 0.15 0.01 −0.16 0.01 −0.04 0.52 0.10 0.11 0.20 0.00 0.06 0.30 −0.02 0.73 −0.15 0.02 0.13 0.04

Leptin

Glucagon

0.26 <0.00

−0.03 0.60 −0.07 0.25 −0.06 0.31 0.04 0.54 0.08 0.20 −0.16 0.01 −0.12 0.09 0.12 0.04 −0.15 0.02 −0.23 0.00

T3

0.19 0.00 0.14 0.01 0.19 0.00 −0.00 0.96 −0.25 <0.00 0.18 0.00 0.01 0.91 −0.01 0.80 0.05 0.46 0.20 0.00 0.12 0.04

T4

Table 2. Correlation coefficient (Pr > F) of BW, BW gain (BWG), feed consumption (FC), feed conversion ratio (FCR), phytate P bioavailability (PBA), insulin-like growth factor (IGF)-I, IGF-II, leptin, triiodothyronine (T3), thyroxine (T4), insulin, and glucagon levels in a chicken population divergently selected for low (below diagonal) and high (above diagonal) PBA1

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1

Table 3. The best-fit equation describing hormones affecting phytate P bioavailability (PBA), BW, BW gain (BWG), feed consumption (FC), and feed conversion ratio (FCR) in chicken populations divergently selected for high and low PBA R2

Line High PBA line   PBA = 1.65 + 0.13T3 − 0.06T4   FC = 94.00 + 0.12IGF-II + 0.01INS − 3.93LEP + 1.47T4   BW = 219.90 + 0.41IGF-II + 3.82T4   BWG = 43.65 + 0.14IGF-II Low PBA line   PBA = 0.26 − 0.02IGF-I − 0.22T3   FC = 73.67 + 0.47IGFI + 0.18IG-FII + 0.19GLU + 5.18T3   FCR = 0.80 + 0.01LEP + 0.02 T3   BW = 219.50 + 0.93IGF-I + 5.29LEP   BWG = 28.75 + 0.11IGF-II + 1.61LEP + 2.60T3

0.084 0.186 0.070 0.084 0.097 0.138 0.058 0.058 0.106

1 IGF = insulin like growth factor; LEP = leptin; INS = insulin; GLU = glucagon; T3 = triiodothyronine; T4 = thyroxine.

low PBA line (Table 2), but no such relationship exists in the high PBA line. Even though there were no line differences for T4 level, there was a significant negative correlation between PBA and T4 in the high PBA line. The blood levels of thyroid hormones could be an indicator of a chicken’s ability to retain phytate P. The genes in the thyroid hormone pathway may be key in the identification of genes associated with PBA. There was a line difference for glucagon level (Table 1). Glucagon has been reported to stimulate calcitonin release and it induces hypophosphatemia (Srivastav and Swarup, 1983). However, there were no significant correlations between glucagon and PBA in both high and low PBA lines, suggesting that the line differences in glucagon may be due to factors other than PBA. Glucagon is correlated with BW, BWG, FC, and FCR in the high PBA line, but not in the low PBA line. However, glucagon contributed significantly to the predictive equation for FC in the low PBA line. Insulin and leptin levels were similar in both lines, and were not correlated with PBA. Insulin, glucagon, and leptin may not have any substantial role in PBA. Kolodziejska and Funk (1926) have reported that insulin plays an insignificant role in total P metabolism in the blood. Insulin was positively correlated with FCR in the high PBA line, whereas in the low PBA line, it was negatively correlated with FCR, but positively correlated with BW and BWG. The association of insulin in the high PBA line may be through FC, rather than BW, because insulin contributed positively to the predictive equation for FC, but not for BWG. A role for leptin in mineral metabolism has not been reported for other species. McMurtry et al. (2003) observed that chicks fed low P diets had elevated plasma leptin levels. However, in this study leptin levels were the same for high and low PBA lines. Leptin, on the other hand, contributed toward the prediction of FCR, BW, and BWG in the low PBA line. The results of this study indicate that the physiology of the divergent lines may be different. Glucagon and IGF-I have a different relation with FCR in the high PBA line compared with the low PBA line. In the high PBA line, T4 is positively correlated with BW, whereas

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onstrated a strong positive association between P intake with plasma IGF-I levels (Giovannucci et al., 2003; Rogers et al., 2005). Kanatani et al. (2002) suggested that high P concentration could stimulate DNA synthesis of osteoblastic MC3T3 cells through increased secretion of immunoreactive IGF-I. Additionally, IGFI has been shown to stimulate in vitro production of 1,25-(OH)2D3 in kidney cells (Condamine et al., 1994). Hypophysectomy abolished an increase in serum 1,25OH2D3 induced by restricted dietary P in rats, but IGF-I restored the increase in serum 1,25-(OH)2D3 induced by restriction of dietary P (Halloran and Spencer, 1988). It is possible that the high IGF-I levels in the high PBA line compared with the low PBA line were because of greater plasma P levels in the high PBA line. Even though there was a significant line difference in IGF-I concentrations, there was a weak negative relationship between IGF-I and PBA in the low PBA line and no relationship in the high line. This suggests that the relationship between PBA and IGF-I may be indirect. When all hormones were considered together, the predictive model indicated that PBA was affected by both T3 and IGF-I in the low PBA line, and by T3 and T4 in the high PBA line (Table 3). Triiodothyronine concentrations were significantly greater in the high PBA line compared with the low PBA line (Table 1). The thyroid gland secretes T3, T4, and calcitonin, which affect numerous metabolic processes including Ca and P homeostasis. Thyroid hormone enhances 1,25-(OH)2D3 action in the small intestine (Cross and Peterlik, 1991), and consequently increases P absorption from the intestinal tract through the stimulation of the secondary active, sodium-coupled phosphate co-transport system in the small intestine (Schroder et al., 1996). Chicks fed a P-deficient diet have reduced blood T3 levels compared with their control counterparts (Parmer et al., 1987). Severe P deficiency affects thyroid hormone function by decreasing serum T3 levels. Therefore, the reduced ability of the low PBA line to utilize phytate P could reflect the lower T3 level in the low PBA line compared with the high PBA line. It is worth noting that there is a significant negative correlation between PBA and T3 in the

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in the low PBA line, it was T3 that was correlated with BW. It is apparent that hormone dynamics are associated with PBA in poultry, and thyroid hormone levels could be an indirect predictor of a bird’s ability to utilize phytate P.

ACKNOWLEDGMENTS The authors thank Cheryl Pearson Gresham (Department of Poultry Science, University of Georgia, Athens) for her technical assistance and Donna Brocht (USDA Animal Biosciences and Biotechnology Laboratory, Beltsville, MD) for performing the hormone assays.

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