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Poult Sci 2007. 86:2343-2349. doi:10.3382/ps.2007-00110
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METABOLISM AND NUTRITION

The Effects of In Ovo Feeding Arginine, β-Hydroxy-β-Methyl-Butyrate, and Protein on Jejunal Digestive and Absorptive Activity in Embryonic and Neonatal Turkey Poults

O. T. Foye*,1, P. R. Ferket* and Z. Uni{dagger}

* Department of Poultry Science, North Carolina State University, Raleigh 27695-7608; and {dagger} Faculty of Agricultural, Food and Environmental Quality Sciences, Department of Animal Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel

1 Corresponding author: Ondulla.Foye-Jackson{at}ars.usda.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In ovo feeding, injecting nutrients into the amnion of the avian embryo, may enhance jejunal nutrient uptake, activity of the intestinal enzymes, and posthatch growth. This hypothesis was tested in the following in ovo feeding (IOF) experiments. In experiment 1, 400 eggs were evenly distributed among 4 nutritional treatments at 23 d of embryonic development (23E) and administered 1 of 4 treatments as a 2 x 2 factorial arrangement of arginine (ARG 0, 0.7%) and β-hydroxy-β-methyl-butyrate (HMB 0, 0.1%). Tissues were assayed for maltase, sucrase, and leucine aminopeptidase (LAP) at 25E, hatch, and 3, 7, and 14 d. In experiment 2, all IOF procedures were repeated and treatments were administered at 21E: injected or noninjected control, 21% egg white protein (EWP), 21% EWP + 0.1% HMB. In experiment 3, two hundred eggs were evenly distributed among the following treatments at 23E: noninjected control or 0.7% ARG + 0.1% HMB + 21% EWP. Jejunal samples were assayed for glucose or alanine uptake at 23E, 25E, and hatch (experiment 2), and hatch and 7 d (experiment 3), respectively. All poults were fed a turkey starter diet ad libitum immediately upon hatching. There was a highly significant HMB x ARG interaction on jejunal sucrase, maltase, and LAP activities at 25E and 14 d. Poults in ovo (IO) fed HMB + ARG had approximately a 2- to 3-fold increase in jejunal sucrase, maltase, and LAP activities at 25E, and a 3-fold increase at 14 d, over other treatments. Poults IO fed EWP + HMB (experiment 2) had enhanced glucose uptake at 25E, whereas poults IO fed ARG + HMB + EWP (experiment 3) had enhanced alanine uptake at hatch and 7 d. These studies demonstrate that IOF ARG, HMB, and EWP may enhance jejunal nutrient uptake and digestion in turkeys.

Key Words: in ovo feeding • jejunal • digestion • absorption • turkey


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Precocial birds hatch with an immature gastrointestinal tract (Klasing, 1998). During the first 72 h posthatch, the gastrointestinal tract undergoes rapid morphological, biochemical, and cellular development to better digest and absorb orally consumed nutrients (Uni et al., 1996, 1998, 1999, 2000, 2003). Thus, young hatchlings may experience suboptimal nutrient digestion and absorption during this window of time in which the gut is reaching functional maturity. It has been hypothesized that low digestive and absorptive capacity of the gut (Klasing, 1998) may limit the energy and nutrients to the chick (Kirkwood, 1983; Lilja, 1983; Kirkwood and Prescott, 1984; Konarzewski et al., 1989), and delay subsequent early posthatch growth. Consequently, limited hepatic glycogen and residual yolk energy reserves (Nir and Levanon, 1993) are rapidly depleted (Rosebrough et al., 1978; John et al., 1988; Christensen et al., 2001) during this critical posthatch period.

The oral consumption of nutrients has been shown to accelerate enteric development and function, seen as marked increases in the size and surface area of the intestinal villi (Klasing, 1998; Uni et al., 1998; Geyra et al., 2001). Therefore, early feeding (immediate access to feed upon hatching) may accelerate gut development and ultimately functional maturity. However, under standard hatchery practices, the avian neonate may be denied access to feed up to 72 h posthatch (Moran and Reinhart, 1980), which may hinder early gut maturation and function. Studies have shown that poults denied access to feed 48 h post-hatch have reduced absorptive surface area, number of cells/crypt, percentage of proliferating cells (Geyra et al., 2001), and the transcriptional factors needed for expression of intestinal genes responsible for digestion and absorption (Geyra et al., 2002). Consequently, denied or delayed access to feed may developmentally delay post-hatch enteric maturation (Geyra et al., 2001, 2002) and limit subsequent posthatch growth (Careghi et al., 2005).

We hypothesize that in ovo feeding (IOF; Uni and Ferket, 2003), the administration of exogenous nutrients into the amnion of the late-term avian embryo, may serve as a tool to overcome growth constraints imposed by limited digestive capacity in hatchlings by enhancing intestinal function and maturation prior to hatching. Naturally, the avian embryo orally consumes the amniotic fluid prior to pipping the air cell, thus consuming the supplemented nutrients that are presented to the enteric tissues, and may stimulate nutrient digestion and absorption by upregulating the activity of the nutrient transporters and brush border enzymes prior to hatching. Hence, the in ovo (IO) fed avian neonate may have a greater capacity to digest and absorb nutrients from an exogenous diet relative to the conventional hatchling. Previous IOF experiments (Tako et al., 2004) demonstrated that poults IO fed the leucine metabolite, β-hydroxy-β-methyl-butyrate (HMB) had a 45% increase in jejunal villus surface area at hatch in comparison with the controls, whereas IOF carbohydrate alone or in combination with HMB resulted in a 33% increase in the villus surface area at 3 d posthatch in comparison with the controls. Therefore, we aim to elucidate the effects of IOF HMB, the leucine metabolite, and arginine on the activity of the jejunal brush border enzymes, sucrase, maltase, and leucine aminopeptidase (LAP) in the late term embryo (25 d of embryonic development), hatchling, and posthatch poult (3, 7, and 14 d). Arginine was chosen as an IOF component due to reports demonstrating physiological enhancements with arginine supplementation (Chevalley et al., 1998; Kita et al., 2002; Flakoll et al., 2004; Kim et al., 2004).

Numerous studies have shown that increased dietary carbohydrate intake up-regulates jejunal glucose uptake (Diamond et al., 1984; Diamond and Karasov, 1987; Ferraris and Diamond, 1993; Ferraris, 2001). However, in this study (experiment 2) we aim to determine the effect of IOF nonspecific dietary substrates such as egg white protein alone or with HMB on jejunal glucose uptake in embryonic poults and hatchlings. Egg white protein was chosen as an ideal protein source due to its predominance within the egg. In addition, we aimed to determine the effect of IOF saline on jejunal sodium glucose cotransport activity because sodium ions are utilized for intestinal glucose transport. Jejunal glucose transport assays were performed on tissues harvested from turkey embryos and new hatchlings in an effort to better determine the effects of IOF saline, egg white protein alone or with HMB on jejunal glucose uptake, without the influence of a high carbohydrate diet typically found in the turkey starter diet.

Several preliminary IOF studies conducted in our lab (O. T. Foye, unpublished data) demonstrated that 1.5 mL of 0.4% saline had no effect on jejunal alanine uptake in turkey embryos or poults. Hence, the noninjected control was chosen for comparisons in this trial in which we aim to determine the effect of IOF dietary egg white protein, arginine, and HMB on jejunal alanine uptake in turkey hatchlings. Also, repeated experiments within our lab (O. T. Foye, unpublished data) demonstrated that IOF of egg white protein, arginine, and HMB had no effect on jejunal alanine transport activity in avian embryos; therefore, measurements were taken at hatch and 7 d posthatch in trial 3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Incubation and In Ovo Feeding
In experiments 1, 2 and 3, viable Hybrid turkey embryos were obtained at 19 d of incubation from a commercial hatchery (Prestage Farms, Clinton NC), and incubated according to standard hatchery practices (99.9 to 100.0°F). In experiment 1, 400 eggs were evenly distributed among 4 to 5 g weight categories ranging from 65 to 85 g per egg at 21 d of incubation (21E). These eggs were evenly distributed among 4 treatment groups of 100 eggs each, such that the weight distribution profile among 4 treatment groups was identical. At 23 d of incubation (23E), each egg was candled to identify the location of the amnion. A hole was incised using a 23-gauge needle, and 1.5 mL of IOF solution was injected into the amnion using a 23-gauge needle to a depth of about 15 mm. The injection site was disinfected with ethyl alcohol, sealed with cellophane tape and transferred to hatching baskets. In experiment 1, the IOF solutions were the following: 0.1% HMB (calcium salt provided by Metabolic Technologies Inc., Ames, IA) in 0.4% saline (HMB); 0.7% arginine in 0.4% saline (ARG), 0.1% HMB + 0.7% ARG in 0.4% saline (HMB + ARG). Jejunal tissues were assayed for maltase, sucrase, and LAP activity at d 25 of embryonic development (25E), hatch, and 3, 7, and 14 d posthatch in the first experiment. The controls were noninjected but were subjected to the same handling procedures as the IOF treatment groups. Preliminary experimentation (O. T. Foye, unpublished data; Uni and Ferket, 2003) conducted in our laboratory indicated that IOF at a range of 1.0 to 2.0 mL of 0.4 to 0.9% saline did not affect embryo/ poult BW or jejunal brush border activity; therefore, the noninjected controls were chosen for comparisons in this trial.

In experiment 2, 400 eggs were evenly distributed as previously mentioned among 4 treatment groups of 100 eggs each. At 21E, each egg was candled to identify the location of the amnion, and all procedures were repeated as mentioned previously. Each egg/embryo was administered 1.5 mL of the following IOF solutions: 21% egg white protein in 0.4% saline (EWP); 21% EWP + 0.1% HMB in 0.4% saline (EWP + HMB); saline injected and noninjected controls. Jejunal samples were harvested at 23E, 25E, and hatch and analyzed for jejunal glucose uptake by radioisotopic accumulation.

In experiment 3, two hundred eggs were evenly distributed among 2 treatments and administered 1.5 mL of 0.7% ARG + 0.1% HMB + 21% EWP in 0.4% saline or noninjected at 23E. All IOF procedures were repeated as previously mentioned in experiment 1. Jejunal samples were harvested and analyzed at hatch and 1 wk posthatch for jejunal alanine uptake by radioisotopic accumulation.

Animal Husbandry
Upon hatching, each treatment group and poult was identified by neck tag and recorded. Hatchability rate of viable eggs exceeded 96% and did not differ significantly among treatment groups. All poults and treatments were randomly assigned to 1 of 3 rooms (experiment 1) or 1 room (experiment 2 and 3), and provided with supplemental heat to maintain 37°C at North Carolina State University Dearstyne Avian Research Facilities. Each room was bedded with soft pine wood shavings and equipped with automatic drinkers, manual self-feeders, and a supplemental incandescent heat lamp to a spot brooding temperature of 40°C. At hatch, each poult was given immediate ad libitum access to a typical turkey starter diet (2,935 kcal/kg, 27.5% protein, and 5.6% fat) that meets or exceeds the National Research Council (1994) nutrient requirements for turkeys. All experimental protocols were approved by the Institutional Animal Care and Use Committee at North Carolina State University.

Tissue Sampling and Brush Border Enzyme Analysis
In experiment 1, ten birds per treatment were euthanized by cervical dislocation, and within 2 min the jejunum was dissected from the end of the duodenal loop to the Meckel’s diverticulum, flushed with ice-cold saline (0.9%), and placed on ice until storage at –20°C for future analysis. Jejunal samples were obtained at 25E, hatch, and 3, 7, and 14 d posthatch. In experiment 1, jejunal samples were analyzed for sucrase and maltase activities using modified methods described by Dahlqvist (1968), and LAP activity was determined using methods described by Goldbarg et al. (1959). The jejunal samples were homogenized in ice cold saline (5 mg/5 mL of tissue) using a polytron homogenizer. The supernatant from each sample was diluted (1:50 for analysis of sucrase and maltase activity; 1:75 dilution for LAP analysis), and 25 µL was pipetted in triplicate in a 96-well plate. Subsequently, 25 µL of substrate (50 mM maltose solution for the maltase assay or 56 mM sucrose solution for the sucrase assay) was added to the wells in duplicate. Twenty-five microliters of distilled water was added to a separate corresponding well to serve as the tissue blank, and each 96-well plate was incubated at 37°C for 30 min. At the end of this incubation period, 200 µL of enzyme-color reagent was added to all wells. [Enzyme solution = 1 purpurogalin glucose oxidase capsule (Sigma # 510-6, St. Louis, MO) dissolved in 100 mL of distilled water in an amber bottle. Color reagent = 50 mg of o-dianisidine dihydrochloride (Sigma #510-50) dissolved in 20 mL of distilled water. The enzyme-color reagent was prepared by combining 100 mL of the enzyme solution with 1.6 mL of the color reagent.] Each plate was then incubated at 37°C for 30 min and read using a microplate plate reader at a wavelength of 450 nm. The quantity of substrate hydrolyzed was determined by colorimetric analysis, and unknown values of maltase and sucrase activity were determined using a standard curve.

Protein Determination
Protein was determined using the Lowry method as described by BioRad Protein Assay kit with Bovine Serum Albumin Assay Standard II BioRad #500-0007, BioRad #500-0115, BioRad #500-0114, and BioRad #500-0113 reagents.

Jejunal Nutrient Uptake Analysis
Reagent Preparation.
Stock solutions of 40 mM L-alanine, 40 mM {alpha}-methyl-L-glucose, 200 mM glutamine, and 40 mM butyrate were prepared and frozen in small aliquots. Transport buffer (pH 7.4) was prepared by dissolving the following compounds: (8.18 g of NaCl, 0.36 g of KCl, 0.37 g of CaCl2, 0.16 g of KH2PO4, 0.30 g of MgSO4, 5.96 g of HEPES) in 1 L of distilled water and stored at 4°C for 1 to 2 mo. On the day of the assay, an appropriate volume (10 mL/bird/transport assay) of preassay transport buffer (TB) was prepared with a final concentration of 2.5 mM glutamine and 0.5 mM butyrate. Subsequently, working alanine or glucose TB was prepared using an appropriate volume (8 mL/intestine/ transport assay) of the preassay TB. L-Alanine and {alpha}-methyl-L-glucose was supplemented to the working alanine and glucose TB with a final concentration of 0.80 mM L-alanine (alanine transport activity) or {alpha}-methyl- L-glucose (glucose transport activity). Subsequently, 1.5 µCi of either C14 methyl-L-glucose and H3 methyl-L-glucose (glucose transport) or H3 L-alanine (alanine transport) was supplemented to 100 mL of appropriate working alanine or glucose TB. The C14 methyl-L-glucose was used to measure active glucose transport, whereas H3 methyl-L-glucose measured passive glucose uptake.

A set of 4 plastic 5-mL beakers was labeled for the alanine transport assay, whereas a set of 3 beakers was labeled for the glucose transport assay. One set of beakers for each assay (glucose transport and alanine transport assay) was labeled for the preassay incubation at 37°C for 10 min with 2.0 mL of preassay TB/bird. One set of beakers was labeled for 2.0 mL of working assay TB, and the second set of beakers was labeled for 2.0 mL of 2.5% trichloroacetic acid (TCA). A set of 10 mL polystyrene centrifuge tubes were labeled and placed into test tube racks (4 tubes/intestine for alanine transport activity, 3 tubes/intestine for glucose transport activity). An additional set of beakers (3 beakers /intestine) was labeled, and 2.0 mL of working alanine assay TB buffer was aliquoted to the appropriate beaker for the alanine transport assay conducted at 4°C, to measure passive alanine uptake.

Transport Assay Procedure.
At the time of sampling, 20 birds per treatment were euthanized by cervical dislocation, and the jejunum was dissected from the end of the duodenal loop to the Meckel’s diverticulum, flushed with ice-cold saline, and immediately assayed. One-millimeter jejunal rings were assayed in triplicate. Each ring (3 rings glucose assay; 4 rings alanine assay) was opened and placed in preassay TB. The beakers containing pre-assay TB and jejunal rings and beakers containing the working assay TB were incubated in a 37°C water bath for 10 min with gentle shaking. Each jejunal ring was transferred from the preassay TB to a corresponding beaker containing 2.0 mL of working assay TB at timed intervals of 20 to 30 s with vigorous shaking for 6 min (alanine) and 15 min (glucose). Subsequently, each ring was rinsed in 3.0 mL of ice-cold 5.5% mannitol at intervals of 20 to 30 s and transferred to a corresponding beaker containing 2.0 mL of TCA to stop transport activity. In the alanine transport assay, this entire transfer procedure was repeated at 4°C using 1 piece of intestinal tissue from each poult and was considered an ice value. The ice value was a measure of passive H3 L-alanine uptake and used to correct the calculation of active alanine transport activity. The TCA from each beaker was centrifuged in polystyrene tubes for 5 min at 500 x g. One milliliter of the supernatant and 10 mL of EcoLite scintillation fluid (ICN, catalog # ICN88247505) were added to a scintillation vial, mixed, and counted in a Packard Tricarb 3000 liquid scintillation counter. Specific activity of the buffer was determined by counting 20-µL aliquots of assay buffer and 980 µL of TCA. All vials were counted for 5 to 10 min in a scintillation counter.

Transport Assay Calculations.
The calibration factor was the triplicate disintegrations per minute average (10 mL of scintillation fluid + 980 µL of TCA + 20 µL of assay buffer) divided by 16 (nmol of substrate/20 µL) x 2 (only half of TCA extracted). The disintegrations per minute for each jejunal ring were multiplied by the calibration factor to determine nanomoles of alanine or {alpha}-methyl-L-glucose per sample. The values were divided by the assay times (6 min of alanine transport; 15 min of glucose transport), which yields the nanomoles of isotope/minute/ millimeter. To determine the concentration of alanine specifically transported, the values calculated from the 4°C procedure were subtracted from the values calculated from the 37°C procedure. Within each triplicate, values that were 85% or closer to one another were averaged.

Statistical Analysis
In experiment 1, all data were statistically analyzed using GLM procedures for ANOVA (SAS, 1996). Each bird served as an experimental unit for statistical analysis. Because highly significant age effects were observed, the treatment effects were evaluated by neonatal age (i.e., hatch and 3 and 7 d of age). The data from IO treatments HMB, ARG, noninjected controls, and HMB + ARG were analyzed in a 2 x 2 factorial arrangement, with 2 levels of HMB (0 and 0.1%) and 2 levels of ARG (0 and 0.7%). Variables having different F-test were compared using least-squares-means function in SAS (1996), and the treatment effects were considered significant at P < 0.05. In experiment 2, comparisons among the 4 groups were evaluated for significance by 1-way ANOVA (ANOVA) using Statview 4.0. In experiment 3, comparisons of 2 groups utilized the Student’s t-test. In all cases P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In all experiments, hatchability for all treatments was equal to or greater than 96% and mortality rates were less than 1.0% through 14 d posthatch (data not shown). In experiment 1, there was a significant main effect of HMB on BW from hatch to 14 d of age, and this effect increased with age as indicated by a significant age x HMB effect on BW (data not shown, P < 0.05). However, there were no significant HMB x ARG interactions on BW at any of the time points taken.

Poults IO fed HMB + ARG had a 1.7- to 2.2-fold increase in jejunal sucrase activity at 25E (48 h post-amniotic nutrient administration) and a 3-fold increase at 14 d posthatch in comparison to other IOF treatments, with a highly significant HMB x ARG interaction (Table 1Go). Conversely, there were main and independent effects of HMB and ARG on jejunal sucrase activity at hatch, in which poults IO fed ARG alone had greater sucrase activities than poults IO fed solutions containing HMB. This effect was lost by 3 d posthatch. Arginine alone significantly enhanced jejunal sucrase activity at 7 d posthatch, whereas the presence or absence of both HMB + ARG depressed jejunal sucrase activity in 1 wk poults (Table 1Go).


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  		Table 1. Experiment 1—The effects of in ovo feeding (IOF) arginine (ARG) and β-hydroxy-β-methyl-butyrate (HMB) on jejunal sucrase, maltase, and leucine aminopeptidase (LAP) activity in embryonic and posthatch turkey poults1
 
Additionally, poults IO fed HMB +ARG had a 1.7- to 2.5-fold increase in jejunal maltase activity at 25E (48 h post-amniotic nutrient administration) and a 3-fold increase in jejunal maltase activity at 14 d posthatch in comparison with other IOF treatments, with a highly significant HMB x ARG interaction (Table 1Go). However, there were main effects of ARG at hatch on jejunal maltase activity in which the presence of arginine significantly enhanced jejunal maltase activity in comparison to the absence of arginine (Table 1Go). At 3 d posthatch there was a significant HMB x ARG interaction, as poults IO fed HMB alone had depressed jejunal maltase activity in comparison with the other IOF treatments. Inclusion of HMB reduced jejunal maltase activity in comparison with the absence of HMB at 7 d posthatch (Table 1Go).

There was a significant HMB x ARG interaction on jejunal LAP activity at 25E (48 h post-amniotic nutrient administration), hatch, and 14 d posthatch, with poults IO fed HMB + ARG having a 1.8- to 2.8-fold increase in jejunal LAP activity at 25E and a 3.2- to 3.4-fold increase at 14 d posthatch over other IOF treatments (Table 1Go). Also, at hatch poults IO fed HMB + ARG had significantly enhanced jejunal LAP activity in comparison to the other IOF treatments, whereas these effects were absent at 7 d posthatch.

Embryonic poults (25E) IO fed EWP + HMB had greater jejunal glucose uptake than the controls and other IOF treatment, with a 5- to 10-fold increase in glucose uptake 48 post-amniotic nutrient administration (Table 2Go), whereas IOF EWP alone or in combination with HMB had no effect of jejunal glucose uptake at 23E or hatch. Also, IOF saline alone did not alter jejunal glucose uptake at any of the time points measured (Table 2Go) in comparison with the other IOF treatments. However, poults IO fed ARG + HMB + EWP had significantly greater jejunal alanine uptake in comparison with the noninjected controls at hatch and 7 d posthatch (Figure 1Go).


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  		Table 2. Experiment 2—The effects of in ovo feeding (IOF) egg white protein (EWP) and β-hydroxy-β-methyl-butyrate (HMB) on jejunal glucose uptake in embryonic and turkey hatchlings1
 

Figure 1
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  		Figure 1. Experiment 3—The effects of in ovo feeding (IOF) arginine, egg white protein (EWP), and β-hydroxy-β-methyl-butyrate (HMB) on jejunal alanine uptake in turkey hatchlings. All data represent the mean of 20 sample birds per treatment. The treatments were Noninj = noninjected control; ARG + HMB + EWP = 0.7% arginine + 0.1% HMB + 21% EWP in 0.4% saline. a,bBars within each age with different letters are significantly different P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During the first week posthatch, young hatchlings undergo a metabolic (Rosebrough et al., 1978, 1979; Elwyn and Bursztein, 1993) and physiological transition (Uni et al., 2000; Geyra et al., 2001; Sklan et al., 2003) from an egg to external feed nutriture. Therefore, rapid intestinal adaptation to an exogenous diet and mature function of the gastrointestinal tract is imperative for the survivability and growth of young hatchlings (Uni et al., 2003; Uni and Ferket, 2004). Early feeding accelerates early gut development and functional maturation in hatchlings (Noy and Sklan, 1998, 1999; Uni et al., 1998). In ovo feeding, which is fundamentally feeding the avian embryo, may circumvent the growth constraints imposed by limited gut function in the avian neonate by the oral consumption of enteric modulators (compounds that stimulate development or metabolism of the cells of the digestive system) administered in the amnionic fluid of the avian embryo, which may enhance the gut capacity to absorb and digest dietary nutrients during late-term embryonic development. β-Hydroxy-β-methyl-butyrate has previously been implicated as an enteric modulator (Uni and Ferket, 2003; Tako et al., 2004) due to the vast improvements seen in early gut development of the HMB IO fed chick in comparison to the conventional hatchling. Additionally, our data imply that HMB + ARG, or in combination with egg white protein, may act as a potent enteric modulator that enhances intestinal disaccharidase and LAP activities or jejunal alanine uptake, respectively.

Numerous studies have demonstrated that the intestinal amino acid (Karasov et al., 1987; Torras-Llort et al., 1998) and glucose transporters (Diamond and Karasov, 1987; Karasov et al., 1987; Solberg and Diamond, 1987; Buddington and Diamond, 1989; Ferraris and Diamond, 1989; Ferraris et al., 1992) are upregulated in the presence of increasing concentrations of their specific dietary substrate(s). Dietary supplementation with protein (Scharrer, 1972), free amino acids (Karasov et al., 1987; Stein et al., 1987), or dipeptides (Ferraris and Diamond, 1989) equally enhance the activity of the jejunal peptide, neutral, and basic amino acid transporters. In parallel, we demonstrate that IOF protein in combination with the free amino acid arginine and the leucine metabolite, HMB, enhances jejunal alanine uptake, which suggest enhanced activity of all intestinal amino acid and peptide transporters. Also, our data parallel studies by Gal-Garber et al. (2003) demonstrating that dietary sodium does not affect the activity of the sodium glucose cotransporter, evident by similar sodium glucose transport activities in the saline injected and noninjected controls of experiment 2 . Interestingly, IOF egg white protein in combination with HMB nonspecifically enhanced jejunal glucose uptake 48 h post-IOF, implying that dietary substrates may not only enhance their substrate specific transporters, but may also enhance the activity of other nutrient transporters within the gut. In conjunction with previous reports (Diamond and Karasov, 1987), our data also demonstrate that intestinal adaptation to diet occurs rapidly (48 h post-IOF), seen as enhanced jejunal glucose transport activity in turkey embryos at 25 d of embryonic development.

This data demonstrates that the IO-fed poult may hatch with a greater intestinal digestive and absorptive capacity than the conventional hatchling. Therefore, the IO-fed poult may have improved nutrient acquisition during the critical posthatch period, which may be correlated with improved growth performance (Smith et al., 1990). Hence, IOF may become a tool that could greatly improve the survivability and hatchability of oviparous species.


   ACKNOWLEDGMENTS
 
This work was supported by Prestage Farms Inc. (Clinton, NC); Institute of Nutrition, University of North Carolina—Chapel Hill; North Carolina Agricultural Foundation, Raleigh; Binational Agricultural Research and Development (BARD) Foundation, and the US Poultry and Egg Foundation. Additionally, the authors would like to thank Annette Israel, Carole Morris, Elad Tako, Dan Moore, Renee Plunske, Anael Santos, Fernanda Santos, and Lizza Macalintal within the Department of Poultry Science, North Carolina State University, Raleigh, for their technical assistance.

Received for publication March 12, 2007. Accepted for publication July 23, 2007.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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