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Poult Sci 2006. 85:2251-2259
© 2006 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Differential Expression of Mitochondrial and Extramitochondrial Proteins in Lymphocytes of Male Broilers with Low and High Feed Efficiency1

K. Lassiter*, C. Ojano-Dirain*, M. Iqbal*, N. R. Pumford*, N. Tinsley*, J. Lay{dagger}, R. Liyanage{dagger}, T. Wing{ddagger}, M. Cooper{ddagger} and W. Bottje*,2

* Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville 72701; {dagger} State-Wide Mass Spectrometry Lab, University of Arkansas, Fayetteville 72701; and {ddagger} Cobb-Vantress, Inc., Siloam Springs, AR 72761

2 Corresponding author: wbottje{at}uark.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies were conducted to investigate relationships between mitochondrial and extramitochondrial protein expression, and protein oxidation in lymphocytes obtained from broilers in which individual feed efficiencies were obtained. Lymphocytes were isolated from male broilers from a single line that were shown to exhibit either low (0.48 ± 0.02, n = 8) or high (0.68 ± 0.01, n = 7) feed efficiency (FE). Western blot analysis showed that, compared with lymphocytes from high FE broilers, lymphocytes from low FE broilers exhibited a) higher amounts of oxidized proteins (protein carbonyls), b) lower amounts of 3 mitochondrial proteins [core I, cyt c 1 (complex III), and ATP synthase (complex V)], and c) higher amounts of 2 proteins [30 S (complex II) and COX II (complex IV)]. Two-dimensional gel electrophoresis revealed that the intensities of 25 protein spots from pooled samples of lymphocytes from high and low FE broilers differed by 5-fold or more. Three of these protein spots were picked from the gel and subjected to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. One protein spot of ~33 kDa was tentatively identified by MALDI-TOF as a fragment of collapsin-2, a component of semaphorin 3D. The results of this study provide further evidence of increased oxidation associated with low FE and further evidence of differential protein expression associated with the phenotypic expression of feed efficiency.

Key Words: feed efficiency • lymphocytes • mitochondria • protein expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In raising an animal to market weight, feed accounts for 50 to 70% of the cost. As a result, feed efficiency (FE, gain to feed) is a very important aspect in commercial animal breeding programs. A 250 to 300% increase in FE and BW was observed when a strain of broilers in 1991 was compared with a control population of broilers bred randomly beginning in 1957 (Havenstein et al., 1994). A later study showed that commercial 2001 birds were more efficient whether fed 1957 or 2001 representative diets compared with random bred broilers from 1957 (Havenstein et al., 2003). However, even in light of these significantly improved traits, large variations between strains and within strains remain (Emmerson, 1997). Differences in the growth and efficiency of broilers may be explained in part by variations in the function and biochemistry of mitochondria (Bottje et al., 2004). Because mitochondria are responsible for producing the majority (about 90%) of the cell’s energy, it is our hypothesis that differences in FE and the growth of broilers may be due to inefficient mitochondrial function.

Recent studies using a variety of tissues suggested that mitochondrial dysfunction exists in low FE birds. Mitochondria from low FE birds exhibited lower electron transport chain complex activity and coupling, and higher production of reactive oxygen species (ROS; Bottje et al., 2002, 2004; Iqbal et al., 2004; Ojano-Dirain et al., 2004; Tinsley et al., 2004). Associated with higher ROS production in mitochondria from low FE birds were observations of higher protein carbonyls, indicative of increased protein oxidation (damage). Thus, higher protein oxidation may contribute to mitochondrial dysfunction in low FE broilers.

The respiratory chain on the inner mitochondrial membrane comprises 5 multi-subunit complexes, namely NADH:ubiquinone reductase (complex I); succinate:ubiquinone reductase (complex II); ubiquinol:cytochrome c reductase (complex III); ferrocytochrome c:oxygen oxido-reductase (complex IV); and ATP synthase (complex V). Electrons move down the respiratory chain from complex I to IV where they are transferred to O2, the final electron acceptor. This movement of electrons is coupled to the movement of protons into the intermembrane space, setting up a proton motive force that drives ATP synthesis (Lehninger et al., 1993). Instead of being completely reduced to water, 2 to 4% of the oxygen used by mitochondria may be partially reduced to ROS (Boveris and Chance, 1973; Chance et al., 1979). Reactive oxygen species include compounds such as superoxide and hydrogen peroxide. Mitochondrial inefficiencies may occur, resulting from electrons leaking from the respiratory chain before they are able to reach the terminal electron acceptor. The formation of ROS in mitochondria makes it a significant source of oxidative stress in the cell (Yu, 1994). If left untreated, the oxidation of essential DNA, lipids, and proteins can lead to more inefficiencies in the mitochondrion and cell, promoting more ROS production.

Using Western analysis, several respiratory chain proteins and a channel protein (e.g., adenine nucleotide translocase 1, ANT1) were shown to be expressed at higher levels in breast muscle (Iqbal et al., 2004) and liver (Iqbal et al., 2005) of low FE broilers. Measuring the level of protein expression in high and low FE birds can also be accomplished using 2-dimensional electrophoresis (2-DE; O’Farrell, 1975). In the first dimension, isoelectric focusing is used to separate proteins by their isoelectric point. Proteins are separated in the second dimension on polyacrylamide gels according to their molecular weight. We hypothesize that increased expression of certain mitochondrial proteins might be a compensatory response aimed at maintaining respiratory chain activity or to overcome the increased protein oxidation in low FE birds (Iqbal et al., 2004).

The intent of the current study was to investigate differences in protein expression in lymphocytes between broilers with low and high FE. Lymphocytes are a convenient source of mitochondria that can be obtained from blood samples. Therefore, in terms of developing a tool to predict feed efficiency, lymphocytes would be a potentially ideal tissue to use in testing for such a marker. Therefore, the objectives of this study were to establish relationships between FE in broilers and a) mitochondrial and extramitochondrial protein expression, and b) protein oxidation in lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Birds and Management
Male broilers with high and low FE (n = 7 to 8 per group) were selected from 100 broiler breeder replacement stock tested for feed efficiency as previously described (Bottje et al., 2002) with the exception that FE was determined between 7 and 8 wk instead of between 6 and 7 wk. Briefly, upon arrival at our facility, broilers were acclimated to single-bird cages for 5 d. The FE was calculated from the increase in BW and net feed consumption for the 49- to 56-d interval. The birds were provided ad libitum access to water and to a diet containing 20.5% protein with 3,280 kcal/kg of energy. The birds were color-coded and biochemical analyses were conducted blind; that is, FE values were not revealed until completion of the proposed experiments.

Lymphocyte Isolation and Preparation of Homogenate
Birds were randomly selected and blood was drawn from the wing vein into a syringe flushed with a solution of heparinized saline (80 U/mL). Lymphocytes were isolated from whole blood using density-gradient Histopaque-1077 (a solution of polysucrose and sodium diatrizoate adjusted to a density of 1.077 ± 0.001 g/mL; Sigma Chemical Co., St. Louis, MO) as described by Lassiter (2005). Briefly, whole blood was carefully layered onto an equal volume of Histopaque-1077 in conical centrifuge tubes and centrifuged (400 x g for 30 min) at room temperature to separate the lymphocytes from plasma and erythrocytes. The lymphocyte layer was transferred to a separate conical centrifuge tube, washed with PBS, and centrifuged (250 x g for 10 min) 3 times to remove erythrocytes and platelets. After washing, the pellet was resuspended in 0.5 to 1.0 mL of PBS. To verify the isolation of lymphocytes, blood smears were made and the slides stained with Wright stain and examined under a light microscope (Figure 1Go). To disrupt cellular and mitochondrial membranes, the lymphocyte homogenate was frozen and thawed 5 times in liquid nitrogen. Protein concentrations of each sample were measured using a protein assay kit (Sigma #A-610).


Figure 1
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  		Figure 1. Visualization of a blood smear using light microscopy and Wright’s stain to verify the isolation of lymphocytes from broilers. Arrows: A = lymphocytes, B = contaminant erythrocytes, and C = cells that lysed during the microscope slide preparation.

 
SDS-PAGE and Western Blots
Mini SDS-PAGE and Western blots were used for the detection of respiratory chain protein subunit expression and protein oxidation (carbonyl) in lymphocyte homogenate as previously described (Iqbal et al., 2004). Equal amounts of protein were loaded onto 10% polyacrylamide gels, separated by SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF, Pall Corp., Pensacola, FL) membranes (0.45 µm) in a submerged system using Hoefer transfer units (Hoefer, San Francisco, CA). Briefly, proteins were separated with a 10% polyacrylamide gel in Tris-HCl buffer using a Hoefer electrophoresis mini-gel system at 100 V for 20 min and 200 V for 1 h. After electrophoresis, gels were soaked in transfer buffer (120 mM glycine, 15 mM Tris, and 20% (vol/vol) methanol, pH 8.3) and electroblotted onto PVDF membranes at 80 V overnight followed by 100 V for 1 h (Pumford et al., 1990).

Immunoblots for Mitochondrial Proteins
Blots were developed using specific primary antibodies for respiratory chain proteins using a peroxidase-based chemiluminescence detection system (Iqbal et al., 2004). Blocked blots were incubated at room temperature for 2 h or overnight with primary antibodies diluted in buffer containing 0.5% casein, 150 mM NaCl, 10 mM Tris, and 0.02% sodium ethylmercurithiosalicylate (thimerosal, pH 7.6). Several primary antibodies were used for the detection of respiratory chain proteins (antibody dilution): 30S (1:400), 70S (1:4,000), core I (1:10,000), core II (1:10,000), cyt c1 (1:10,000), ISP (1:10,000), COX II (1:600), and {alpha}-ATP synthase (1:4,000). The 30S, 70S, and {alpha}-ATP synthase antibodies were purchased from Molecular Probes, Inc., Eugene, OR; the COX II antibody was a gift from R. Doolittle, UCSD, San Diego, CA; and the others were a gift from L. Yu, Oklahoma State University, Stillwater.

Following incubation with primary antibodies, blots were washed with detergent buffer (0.5% casein, 150 mM NaCl, 10 mM Tris, 0.02% thimerosal, 0.1% SDS, 5% Triton X-100) and twice with washing buffer (0.5% casein, 150 mM NaCl, 10 mM Tris, 0.02% thimerosal) for 5 min each, and rinsed with distilled deionized H2O (3 times) before and after incubation with detergent or washing buffers. Blots were incubated for 90 min with the appropriate peroxidase-labeled secondary antibodies [goat antimouse IgG (Life Technologies, Gaithersburg, MD) or rabbit antisera (Dako Corp., Carpinteria, CA)] and then washed with detergent, washing buffers, and Tris-saline. Blots were treated with substrate (Supersignal West Dura Extended Duration, Pierce, Rockford, IL) for 5 min and chemiluminescence bands were detected using a charge-coupled device (CCD) camera (LAS 1000 plus, Fuji Photo Co. Ltd., Tokyo, Japan). Bands were quantified using Scion computer software (http://www.scioncorp.com). The molecular weights of separated proteins were estimated by comparison with Prosieve color molecular weight standards (Bio-Whittaker Molecular Applications, Rockland, ME) run in parallel. For internal controls, mouse anti-glyceraldehyde-3 phosphate dehydrogenase (GAPDH) or glutamate dehydrogenase (Chemicon International, Temecula, CA) antibodies were used. Therefore, the values of the mitochondrial protein expression were calculated as the ratio of mitochondrial protein intensity to the GAPDH staining intensity. A representative blot of mitochondrial protein staining and GAPDH staining are provided in Figure 2Go.


Figure 2
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  		Figure 2. Representative Western blots illustrating A) mitochondrial protein staining of complex II subunit 70 and B) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining. Lanes marked L and H indicate samples from broilers with low and high feed efficiency, respectively; MWM indicates the molecular weight markers (which are not clearly visible because the blot image was developed using a chemiluminescence detection system).

 
Immunoblots for Protein Carbonyls
Protein carbonyl levels were determined by Western analysis using a reaction of dinitrophenyl hydrazine with carbonyls (aldehydes and ketones) on oxidized proteins (Keller et al., 1993) in a modified format (Iqbal et al., 2004). Proteins were separated by SDS-PAGE (see above), transferred onto PVDF membranes, and incubated in 10 mL of 20 mM 2,4-(dinitrophenyl hydrazine) in 10% (vol/vol) trifluoroacetic acid and 20 mL of 12% SDS. After 15 min, 30 mL of 2 M Tris-base was added and further incubated for 20 min. Blots were developed as described above for Western blots using anti-dinitrophenyl hydrazine antiserum (1:5,000 dilution, Sigma) with a peroxidase-based chemiluminescence detection system. Similarly, GAPDH was used as internal control as described above.

2-DE
Two-dimensional gel electrophoresis was used to qualitatively determine global differences in expression of proteins in the lymphocyte homogenate prepared from high and low FE broilers. Lymphocyte suspensions from high and low FE groups were pooled (normalized for protein concentration; n = 5 to 6). Lymphocytes were treated with lysis buffer (8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 2% vol/vol immobilized pH gradient (IPG) buffer, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA) and diluted in rehydration buffer (8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 2% vol/vol IPG buffer (pH 3 to 10 nonlinear), 0.33 mg/mL of dithiothreitol, and 2 mg of bromophenol blue). Proteins were separated by isoelectric point using Immobiline DryStrips (pH 3 to 10 nonlinear, 7 cm) that were run on the IPGphor isoelectric focusing system (first dimension, Amersham Biosciences, San Francisco, CA). The IPG strips were incubated in equilibration buffer (50 mM Tris-base, 6 M urea, 30% vol/vol glycerol, 2% wt/vol SDS, a trace of bromophenol blue) containing dithiothreitol and iodoacetamide. The second dimension step was performed on 12% polyacrylamide gels (as previously described) to separate proteins by molecular weight. Proteins on the gels were visualized using silver stain (Invitrogen Corp., Carlsbad, CA). The gels were scanned using the Agfa (Arcus II) scanner to obtain a digital image to be used for analysis. The intensity of the protein spots on the gels was quantified using Melanie 5 software (Bruker Daltonics Inc., Billerica, MA). The relative intensity (% intensity), as described in the Melanie 5 software user manual, was used to normalize spots. This spot normalization is an internal calibration that makes the data independent from experimental variations such as protein loading and staining.

In-Gel Trypsin Digestion
Three protein spots (identified as numbers 18, 19, and 22 with the Melanie 5 software) that were large enough to be cut out manually and showed at least a 5-fold difference in the expression of lymphocyte proteins between groups of high and low FE broilers were subjected to in-gel trypsin digestion using methods described by Li (2000). The protein spots of interest were excised from the gels for mass spectrometry analysis. Briefly, gel pieces were destained using 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. After destaining, the gel pieces were soaked in 0.2 M ammonium bicarbonate before being covered in 100% acetonitrile. Once the gel pieces were dried, up to 400 ng of trypsin (Promega Corp., Madison, WI) in 25 mM ammonium bicarbonate was added to the gel pieces and allowed to incubate overnight at 37°C in a closed incubator. The digests were repeatedly passed through Millipore C18 ZipTip pipette tips (Millipore Corp., Billerica, MA) to remove salts and concentrate the peptides in the digests.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry and Protein Database Search
After desalting and concentrating, the peptide digests were run on the Bruker Reflex III matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics Inc.) equipped with a Scout 384 ion source at the University of Arkansas StateWide Mass Spectrometry Facility. The list of peptide masses generated for the protein spots of interest was used to search the Swiss-Protein database using Protein Prospector on the ExPASy Proteomics Server (http://www.expasy.org). Results from the protein database search were then used to determine the identity of the differentially expressed unknown proteins excised from the 2-DE gel.

Statistical Analyses
Data for immunoblots are presented as the mean ± standard error. The means were compared by t-test using JMP IN statistical analysis software (SAS Institute Inc., Cary, NC). A probability level of P ≤ 0.05 was considered statistically significant unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The growth performance data of broilers used in this study are presented in Table 1Go. Feed efficiency (g of gain/g of feed) was 0.68 ± 0.01 for high FE broilers compared with 0.48 ± 0.02 for low FE broilers. Although there was no difference in feed intake, high FE broilers gained more weight than did low FE broilers. Feed conversion ratios were also significantly different between high and low FE groups (Table 1Go).


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  		Table 1. Growth performance (mean ± SE) of broilers with high and low feed efficiency (wk 8)
 
Western analysis was conducted to assess expression of several respiratory chain proteins and protein oxidation in lymphocytes obtained from broilers with low and high FE. Expression of respiratory chain proteins detected in this study is shown in Figure 3Go. These proteins included 2 in complex II [30S and 70S (nuclear DNA-encoded subunits], 4 proteins in complex III [core I, core II, cyt c1, ISP (nuclear DNA-encoded subunits)], 1 protein in complex IV [COX II (mitochondrial DNA-encoded subunit)], and 1 protein in complex V [{alpha}-ATP synthase (nuclear DNA-encoded subunit)]. Although we obtained good cross-reactivity of several proteins in complex I in other tissues, there was no immunoreaction with any complex I antibodies that were tested in lymphocytes. Although 2 proteins (30S of complex II and COX II of complex IV) were higher (P < 0.05) in lymphocytes from low FE broilers, 3 other proteins (core I and cyt c1 of complex III and {alpha}-ATP synthase subunit of complex V) were higher in lymphocytes from high FE broilers. There were no differences (P > 0.05) in expression of 3 other proteins (core II and ISP of complex IV and the 70S subunit of complex II) between lymphocytes from high and low FE broilers. The level of protein carbonyls (an indicator of protein oxidation) was 13% higher in the lymphocytes isolated from low FE broilers compared with those from high FE broilers (Figure 4Go).


Figure 3
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  		Figure 3. Expression of electron transport chain protein subunits in lymphocytes from broilers with low and high feed efficiency (FE). Each bar represents the mean ± standard error for high (black bar, n = 6) and low FE (white bar, n = 8). Mt- and n- represent mitochondrial- or nuclear-encoded protein subunits, respectively. *P ≤ 0.05, **P ≤ 0.01.

 

Figure 4
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  		Figure 4. Level of protein carbonyls in lymphocytes of broilers with low and high feed efficiency (FE). Each bar represents the mean ± SE for high FE (black bar, n = 5) and low FE (white bar, n = 8). *P ≤ 0.05.

 
Two-dimensional electrophoresis of pooled lymphocyte samples revealed several protein spots (Figure 5AGo) that differed between high and low FE groups. In this study, only those proteins that showed at least a 5-fold difference in expression were presented (Figure 5BGo). Using these criteria, it was determined that qualitatively, 17 proteins were higher in lymphocytes from low FE broilers (negative values) and 8 proteins were higher in lymphocytes from high FE broilers (positive values). It should be pointed out that we are at the initial stages of 2-DE analysis in our FE studies. Therefore, the fold differences of the differentially expressed proteins may change in subsequent experiments.


Figure 5
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  		Figure 5. Two-dimensional gel electrophoresis (A) and comparison of pooled protein spots differing by 5-fold or more (B) from lymphocyte homogenate samples pooled on the basis of equal protein (25 µg each) from broilers with low and high feed efficiency (FE). Protein spots 18, 19, and 22 (marked with *) were selected for protein identification. Spot 18 was identified as an unknown lymphocyte protein of the precursor protein pro-semaphorin 3D (collapsin-2) by matrix-assisted laser desorption time-of-flight mass spectrometry analysis.

 
Of the 3 protein spots that were picked from the 2-DE gels and subjected to trypsin digest, only 1 (spot 18 in Figure 5AGo) contained sufficient amounts of protein to generate data with MALDI-TOF mass spectrometry. This problem could be overcome in future studies by running gels in sets of 4 and combining the protein spots of interest from each of the gels into 1 tube for trypsin digestion (our unpublished observations). The excised protein was identified by comparison of the tryptic peptides’ molecular masses determined by MALDI-TOF and the calculated peptide masses from the Swiss-Protein database. A 33-kDa fragment of the precursor protein pro-semaphorin 3D (collapsin-2) was tentatively identified as the protein whose peptide masses best matched those from the unknown (spot 18) lymphocyte protein. Figure 6Go illustrates the amino acid sequence of collapsin-2 and the tryptic fragments of the unknown lymphocyte protein (spot 18) whose masses match those from collapsin-2.


Figure 6
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  		Figure 6. Amino acid sequence of collapsin-2. The amino acids underlined in bold indicate the tryptic fragments of the unknown lymphocyte protein (spot 18) whose masses match those from collapsin-2 (identified by matrix-assisted laser desorption time-of-flight mass spectrometry).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the FE values for high and low FE groups (Table 1Go) were lower than values in previous studies (Bottje et al., 2002; Iqbal et al., 2004, 2005; Ojano-Dirain et al., 2004), the magnitude of difference between the 2 groups (approximately 0.2 units) was similar. Birds in this study were tested for FE 1 wk later than in previous studies, which would account for the heavier BW in the present study. In agreement with the earlier studies (Bottje et al., 2002; Ojano-Dirain et al., 2004; Iqbal et al., 2005), birds in the high FE group gained more weight on the same amount of feed compared with the low FE group.

Because mitochondria are responsible for producing 90% of the energy for the cell, the proteins of the respiratory chain are important for optimal function of the organelle in the production of ATP. Expression of proteins in the respiratory chain are controlled by both nuclear (n) and mitochondrial (mt) DNA (Wei, 1998). Recent studies have shown that the balance between n- and mt-encoded DNA is important for mitochondria to function normally (Nijtmans et al., 2002). However, the balance of this genetic material is affected by oxidation within the mitochondria. Mitochondrial DNA is more vulnerable to oxidative damage than nDNA because it lies in close proximity to the electron transport chain and lacks the protective histones of nDNA (Wei, 1998). Up to 4% of the oxygen consumed by mitochondria in ATP synthesis may be incompletely reduced to ROS such as hydrogen peroxide and superoxide, making the mitochondria a significant source of oxidative stress in the cell. Failure to metabolize ROS by antioxidants results in damage of important structures in the cell that can lead to further oxidative damage and more generation of ROS.

Similar to our earlier findings in a variety of tissues (liver, breast, leg, heart, gut; Bottje et al., 2004), the protein carbonyl levels in lymphocytes were also higher in low FE broilers, indicating increased protein oxidation (damage) in low FE broilers. Thus, protein oxidation is apparently an important biochemical characteristic and hallmark associated with the phenotypic expression of low FE. Oxidative damage to mitochondrial proteins and mtDNA has been identified in cardiac diseases as well as metabolic diseases (Tritschler et al., 1994; Kristal et al., 1997; Wallace, 1999; Castegna et al., 2002a,b; Kang and Hamasaki, 2005). Evidence has shown that oxidative stress can be modulated by caloric restriction in the diet (Lass et al., 1998; Usuki et al., 2004; Sanz et al., 2005). By decreasing the percentage release of ROS per total electron flow, and therefore energy production by the electron transport chain, caloric restriction lowers oxidative stress levels and reduces the rate of aging (Sanz et al., 2005). Decreasing the levels of oxidative stress using caloric restriction has also been reported in broilers (Iqbal et al., 1999). Because low FE broilers from the same genetic line exhibit increased ROS production and increased protein oxidation compared with high FE broilers (Bottje et al., 2002, 2004), mechanisms involved in low FE may be similar to the mechanisms found in aging and other diseases such as Alzheimer’s and Parkinson’s disease (Wei, 1998; Castegna et al., 2002a,b; Winkler-Stuck et al., 2005).

The measurements of respiratory chain protein expression and carbonyls in lymphocytes complement previous studies in breast muscle, liver, heart, and duodenal mitochondria (Bottje et al., 2004; Iqbal et al., 2004; Ojano-Dirain et al., 2004; Tinsley et al., 2004). In the current study, 3 respiratory chain proteins encoded by nDNA (core I, cyt c1, and {alpha}-ATP synthase) were expressed at higher levels in high FE broilers, whereas the expression of COX II, encoded by mtDNA, was lower in the mitochondria of high FE broilers compared with low FE broilers. These observations are opposite to the reports in breast muscle, liver, and heart (Iqbal et al., 2004; Tinsley et al., 2004), but similar to those reported in duodenal mitochondria (Ojano-Dirain et al., 2004). The exact reasons for differential expression of specific mt- and n-DNA encoded proteins in various tissues are not yet known.

After conducting 2-DE on pooled samples of lymphocytes from broilers with high and low FE, 25 protein spots were found to differ qualitatively by 5-fold or more between the 2 groups. One of these proteins was tentatively identified by MALDI-TOF mass spectrometry analysis as a fragment of the precursor protein pro-semaphorin 3D (collapsin-2). Collapsin-2 is a part of a group of proteins known as semaphorins (Adams et al., 1997). When activated by proteolytic cleavage, members of this group of proteins collapse and paralyze the movement of growth cones (Luo et al., 1995; Koppel et al., 1997) that are important to the growth and development of axons (Puschel et al., 1995). Why this protein would be identified in lymphocytes is not known at this time. Currently, there are no commercially available antibodies to semaphorin to confirm its identity in lymphocytes.

Similar to the results obtained from Western blot analysis observed in this study and previous studies (Iqbal et al., 2004, 2005; Ojano-Dirain et al., 2005) in which differences in individual proteins were observed but no consistent trend was apparent, the 2-DE analysis did not indicate that protein levels were predominantly higher or lower in one group or the other. With this observation in mind, it would be advantageous to investigate whether one or more of these proteins have a role in determining feed efficiency. Identification of differentially expressed proteins may give researchers an idea of which cellular or metabolic processes are being affected between high and low FE birds. In addition, we may be able to use 2-DE in combination with mass spectrometry to identify proteins for the possible development of a biomarker used as a tool to predict FE in broilers.

In summary, the results of these studies indicate that the level of protein carbonyls (indicators of protein damage) is higher in the lymphocytes of low FE broilers than in high FE broilers. Similar to previous studies in our lab, Western blots and 2-DE analysis showed that mitochondrial and extramitochondrial proteins were differentially expressed between the 2 groups. Collapsin-2 was tentatively identified following 2-DE and MALDI-TOF, and was higher in lymphocytes from low FE broilers compared with high FE broilers. The findings presented in this study may provide researchers with a better understanding of the relationship between feed efficiency and the expression of mitochondrial and extramitochondrial proteins and may help in developing a biomarker for feed efficiency.


   ACKNOWLEDGMENTS
 
The authors would like to thank L. Yu (Oklahoma State University, Stillwater, OK) for the generous gifts of primary antibodies for complex III. Thanks also go to H. Brandenburger for her help in technical editing of the manuscript. This research was presented at the Poultry Science Association’s annual meeting in St. Louis, MO (July 2004) and Auburn, AL (July 2005).


   FOOTNOTES
 
1 This research was funded by USDA-NRI grant (#2001-03443), and Cobb-Vantress, Inc. to W. Bottje and M. Iqbal, and is published with support by the Director of the Agriculture Research Experiment Station, University of Arkansas, Fayetteville. Back

Received for publication May 18, 2006. Accepted for publication July 26, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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C. Ojano-Dirain, M. Toyomizu, T. Wing, M. Cooper, and W. G. Bottje
Gene Expression in Breast Muscle and Duodenum from Low and High Feed Efficient Broilers
Poult. Sci., February 1, 2007; 86(2): 372 - 381.
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