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The relationship between mitochondrial function and residual feed intake in Angus steers¹

Division of Animal Sciences, University of Missouri, Columbia 65211

	Abstract

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The objective of this study was to examine the relationship between mitochondrial function and residual feed intake in Angus steers. Individual feed intakes were recorded for a contemporary group of 40 steers via the GrowSafe feed intake system. Intakes were then used to calculate residual feed intake (RFI), a measure of efficiency. Based on these calculations, 9 low (RFI = –0.83) and 8 high (RFI = 0.78) RFI animals were selected for further study. Blood samples were collected via jugular venipuncture 1 wk before slaughter for the determination of plasma glucose and insulin concentrations. Tissue samples were taken from the LM from both the high and low RFI animals and mitochondria were isolated for measurement of oxygen consumption and hydrogen peroxide production. Average daily gain and carcass composition were not different between the high and low RFI steers; however, ADFI by the high RFI animals was 1.54 kg/d greater (P < 0.001) than for the low RFI animals. Low RFI steers exhibited a greater (P < 0.05) rate of state 2 and 3 respiration, respiratory control ratio, and hydrogen peroxide production than high RFI steers when provided with glutamate or succinate as a respiratory substrate. The acceptor control and adenosine diphosphate:oxygen ratios were not different between the 2 groups for either substrate. When hydrogen peroxide production was expressed as a ratio to respiration rate there was no difference between groups, signifying that electron leak was similar for both groups. Plasma glucose concentration was greater (P < 0.05) in the high RFI steers than in the low RFI steers; however, plasma insulin concentration was not different (P = 0.22) between the 2 groups. The ratio between plasma glucose and insulin concentration was similar (P = 0.88) between the 2 groups indicating no difference in glucose metabolism. The increased plasma glucose concentration observed in the high RFI steers was presumed to be the result of a greater feed intake by these animals. It seems that mitochondrial function is not different between the high and low RFI groups but rather the rate of mitochondrial respiration is increased in low RFI steers compared with high RFI steers.

Key Words: feed efficiency • mitochondria • residual feed intake

	INTRODUCTION

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Substantial improvements in the profitability of beef production could be made by improving G:F because feed costs represent a major proportion of production costs (Arthur et al., 1996). Dietary manipulations such as balancing the AA to energy ratio of the diet (Mueller et al., 2004) and removing forage from the diet (Willis and Kerley, 2004) can result in improved G:F; however, the variation in G:F among animals remains (Mueller et al., 2004). Animal selection for superior G:F traits is mandatory if improving G:F in beef cattle is to be realized.

Residual feed intake (RFI) is an index that can be used to calculate an animal’s efficiency (Archer et al., 1999) and describes the divergence in intake from that needed for maintenance and growth. Maintenance and growth requirements are not accounted for by G:F, making RFI comparisons between animals a better measure of efficiency. A large genetic variation in RFI exists, and this trait has been found to be moderately heritable (Archer et al., 1998). However, the measurement of individual feed intake, and hence RFI, is difficult and costly. Determining the physiological mechanism that is responsible for the observed differences in feed intake would provide for a more cost-effective method of determining an animal’s RFI status.

Mitochondria are the site of energy production in the cell and produce the majority of cellular ATP. Recent work in poultry (Bottje et al., 2002) and rats (Lutz and Stahly, 2003) has provided evidence of a link between inefficient mitochondrial respiration and decreased G:F. These findings led us to hypothesize that mitochondrial function would be altered similarly in beef cattle. Therefore, our hypothesis was that inefficient mitochondrial respiration was related to decreased G:F in beef cattle. The objectives of this research were to evaluate the relationship between mitochondrial respiration and hydrogen peroxide production in steers selected for low or high RFI.

	MATERIALS AND METHODS

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Animal Management
The protocols using animals in this study were approved by the University of Missouri Animal Care and Use Committee. Forty Angus steers (average initial BW = 325.4 ± 23.7 kg) were used to select high and low RFI animals. The steers were obtained from a single herd enrolled in the MFA Health Track Beef Alliance (Columbia, MO), were all from the same sire, and had been previously vaccinated and preconditioned for 45 d before arrival at the University of Missouri Beef Research Farm.

Upon receiving the animals, electronic ID tags (Allflex USA, Inc., Dallas-Ft. Worth, TX) were attached to the exterior of the left ear for the measurement of individual feed intake with the GrowSafe feed intake system (Model 4000 E, GrowSafe Systems Ltd., Airdrie, AB, Canada). Steers were placed on a receiving diet for 14 d to allow for acclimation to the feeding system. After the acclimation period, the steers were fed Trendsetter SLR (MFA, Inc., Columbia, MO) at a rate of 25% Trend-setter SLR and 75% whole corn until they reached 454 kg. At 454 kg, the diet was switched to 12.5% Trendsetter SLR and 87.5% whole corn for the remainder of the experiment. All steers had ad libitum access to feed and water. The steers were weighed every 21 d, and RFI values were calculated for the entire feeding period. The expected feed intake was calculated by regressing the actual intake against ADG and metabolic mid-weight (Basarab et al., 2003). The RFI value for each animal was calculated as the difference between the actual and expected intake.

Nine low and 8 high RFI steers were selected based on their RFI values and were used for the study of mitochondrial respiration. These 17 steers were transported to the University of Missouri abattoir where the animals were killed to obtain tissue from the LM for mitochondrial isolation. Hot carcass weights were documented for each animal and the carcasses were chilled for 24 h at 5° C. After the 24-h chill, the LM area of each carcass was measured to the nearest 0.01 cm². Subcutaneous fat thickness at the 12th rib was determined using a USDA preliminary yield grade ruler (USDA, 1997) at an anatomical location perpendicular to the vertebral column and ³/3 of the distance caudal to the LM. To determine preliminary yield grades, the fat measurements were then adjusted, correcting for any atypical fat distribution.

Mitochondrial Isolation
Mitochondria were isolated from the LM according to the procedures of Bottje et al. (2002), with modifications. Briefly, 5 to 10 g of tissue was collected and placed in 30 mL of ice-cold medium I (100 mM sucrose, 100 mM Tris-HCl, 46 mM KCl, 10 mM EDTA, pH 7.4). The tissue was minced with scissors, placed back in medium I, and incubated with 8 mg of protease K (Sigma-Aldrich Co., St. Louis, MO) at room temperature for 5 min. The tissue was homogenized (for approximately 2 min) in a Potter-Elvehjem vessel with a Teflon pestle, and then incubated on ice for an additional 5 min. The homogenate was centrifuged at 1,000 x g for 10 min and the pellet containing cellular debris was discarded. The supernatant was then centrifuged at 10,000 x g for 15 min to pellet the mitochondria. The pellet was washed with 10 mL of medium I containing 0.5% BSA. Mitochondria were repelleted by centrifuging at 10,000 x g for 15 min, suspended in 2 mL of medium II (230 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl, 5 mM KH₂PO₄, pH 7.4), and placed on ice until assays were performed. Mitochondrial protein was determined by the Coomassie Plus protein assay kit (Pierce Biotechnology, Inc., Rockford, IL).

Mitochondrial Function Measurement
Oxygen consumption was measured in duplicate with a Clark-type oxygen probe (YSI, Inc., Yellow Spring, OH) in a respiration chamber at 37° C under constant stirring (Bottje et al., 2002). Mitochondria (0.5 mL) were added to 3 mL of respiration buffer (220 mM mannitol, 70 mM sucrose, 3 mM KH₂PO₄, 2 mM HEPES, pH 7.0). Glutamate (10 mM) was used to stimulate respiration at complex I, and succinate (10 mM) was used for complex II respiration. State 3 respiration was initiated by the addition of 10 µL of a 50 mM solution of ADP. The respiratory control ratio was calculated as the ratio of state 3 respiration to state 4 respiration; acceptor control ratio was calculated as the ratio of state 3 to state 2 respiration. The adenosine diphosphate to oxygen consumption ratio (ADP:O) was calculated according to the methods of Eastbrook (1967).

Hydrogen Peroxide Measurement
The production of hydrogen peroxide by mitochondria isolated from steers selected to have a high or low RFI was measured using the procedures of Bottje et al. (2002), with modifications. Hydrogen peroxide was measured using the dichlorofluorescein diacetate probe (Molecular Probes, Inc., Eugene, OR) in a 96-well plate fluorimeter (Fluoroskan Ascent, Thermo Electron Corporation, Vantaa, Finland). Mitochondria (0.05 to 0.1 mg of protein) were incubated with 52 µM of dichlorofluorescein diacetate, 64 µL of buffer (145 mM KCl, 30 mM HEPES, 15 mM KH₂PO₄, 3 mM MgCl, 0.1 mM EGTA, pH 7.4), 10 U of superoxide dismutase, and 10 mM of either glutamate or succinate. Samples were incubated at 37° C for 40 min with fluorescence measured every 5 min. Hydrogen peroxide production was calculated from a standard curve, and was expressed as nanomoles of H₂O₂ generated per minute per milligram of mitochondrial protein.

Plasma Glucose and Insulin
Blood was collected by jugular venipuncture 1 wk before slaughter into Vacutainers containing EDTA as an anticoagulant (Becton, Dickinson and Company, Franklin Lakes, NJ). Samples were collected in the morning before the animal’s first major feeding event. The blood samples were centrifuged at 2,200 x g for 15 min, and the plasma was decanted and frozen at –20° C until further analysis.

Plasma glucose was determined using a colorimetric glucose oxidase kit (Thermo Electron Corporation, Louisville, CO) according to the manufacturer’s instructions. Plasma concentrations of insulin were quantified using a specific, double-antibody, equilibrium radioimmunoassay as described by Elsasser et al. (1986), with some modifications. Preparation of bovine insulin (Sigma-Aldrich Co.) for iodination and for the standard curve was via the method of Sodoyez et al. (1975) for preparation of zinc-free insulin. Ten micrograms of zinc-free bovine insulin was then solubilized in 50 µL of H₂O, combined with 500 µCi of ¹²⁵I-Na, and incubated in the presence of 100 µg of iodogen (Pierce Biotechnology, Inc.) for 6 min with gentle mixing. Recovery of the monoiodinated form of ¹²⁵I-bovine insulin was achieved by differential elution from a 10-mL Sep-Pak C18 cartridge (Mallinckrodt Baker Inc., Phillipsburg, NJ) as previously described by Deleo (1994), as follows. The Sep-Pak C18 cartridge was initially washed with 10 mL of 50% (vol/vol) acetonitrile containing 50 mM tri-ethylamine solution (pH adjusted to pH 3 with phosphoric acid), followed by 10 mL of deionized H₂O before addition of the iodination mixture. The cartridge was then washed sequentially with: 1) 5 mL of 0.4 M phosphate buffer, pH 7.4; 2) 10 mL of 29% (vol/vol) acetonitrile containing 50 mM trimethylamine; 3) 5 mL of 10% (vol/vol) acetonitrile containing 0.2 M ammonium acetate, pH 5.5; and 4) 5 mL of 50% (vol/vol) acetonitrile containing 0.2 M ammonium acetate, pH 5.5. This final fraction was collected and diluted to 25,000 cpm per 100 µL of assay buffer (0.1% gelatin, 0.01 M EDTA, 0.9% NaCl, 0.01 M PO₄, 0.01% sodium azide, 0.1% Tween-20, pH 7.1). Guinea pig antibovine insulin antiserum (Elsasser et al., 1986) was diluted to a final tube dilution of 1:167,000 in assay buffer. Standard concentrations of zinc-free bovine insulin (0.064 to 40 ng/tube) and increasing volumes of a bovine plasma pool (25 to 300 µL) were added to assay tubes in quadruplicate, and the total volume was balanced to 300 µL per tube with assay buffer. All plasma samples (100-µL aliquots) to be analyzed were assayed in triplicate. All components were then incubated at 4° C for 24 h. The antigen-antibody complex was then precipitated following a 15-min, 22° C incubation with 100 µL of a pre-precipitated sheep-anti-guinea pig secondary antibody. The secondary antibody complex was then precipitated by centrifugation at 3,000 x g for 30 min, and the supernatant was discarded by aspiration. Assay tubes containing the precipitate were counted for 1 min on an LKB1275 gamma counter (LKB Wallac, Turku, Finland). Standards and plasma aliquots of the bovine plasma pool were linear (log/logit transformation; r² = 0.98) and parallel over a mass of 0.064 to 40 ng/tube and a plasma volume of 25 to 300 µL. Total specific binding was 38%, the minimum detectable concentration was 0.064 ng/tube, the percentage recovery of mass was 98.1%, and the inter- and intraassay CV were 5.2 and 6.8 %, respectively.

Statistical Analysis
The data were analyzed using the GLM procedure (SAS Inst., Inc., Cary, NC) as a completely randomized design. An alpha level of 0.05 was used for the determination of statistical significance.

	RESULTS AND DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
The growth performance of the high and low RFI steers is shown in Table 1. There were no differences (P = 0.80) in initial or final BW or ADG between the 2 groups. However, G:F was decreased and ADFI was greater (P < 0.001) for the high RFI steers, which consumed 1.54 kg/d more feed than the low RFI steers. Basarab et al. (2003) reported similar data in which ADFI and G:F were greater (P < 0.0001) and ADG and BW of high and low RFI steers were not different. In contrast, poultry researchers (Bottje et al., 2002; Iqbal et al., 2004; Ojano-Dirain et al., 2004) observed increased gain with no difference in feed intake between birds of high and low feed efficiency. The lack of differences in feed intake between high and low feed efficient birds may represent an alternative mechanism that alters G:F in poultry. Carcass composition, as assessed by LM area, s.c. fat thickness over the 12th rib, HCW, and USDA Yield Grade, was not different between the high and low RFI groups. Other investigators (Richardson et al., 2001; Basarab et al., 2003) have reported increased fat deposition in steers selected to have high RFI.

A greater amount of hydrogen peroxide production, which would be indicative of electron leak, would be expected based on the observed lower respiratory control ratio values in high RFI steers in the current study; however, this was not the case. High RFI steers produced less (P < 0.05) hydrogen peroxide than low RFI steers when either glutamate or succinate was provided as a substrate. These observations are in contrast to the observations of Bottje et al. (2002), in which the isolated mitochondria of low feed efficient birds produced greater amounts of hydrogen peroxide. However, because electron leak is a function of respiration (Chance et al., 1979), we expressed H₂O₂ production as a ratio to respiration rate (state 2). We observed no difference between the high and low RFI steers in the amount of electron leak when it was expressed as a function of respiration rate. Increased electron leak, hydrogen peroxide production, or reactive oxygen species would result in the destruction of the mitochondria and ultimately cell death (Fleury et al., 2002), and therefore, most likely would not be involved in altering gain efficiency. Based on these data, mitochondrial function was not impaired in high RFI steers; rather, the flux of electrons through the electron transport chain was impaired.

A reduced supply of substrate to the mitochondria could affect mitochondrial respiration rates. We measured plasma glucose and insulin concentrations as an indicator of glucose metabolism and substrate availability to the mitochondria (Table 3). We observed that high RFI steers had greater (P < 0.05) plasma glucose concentrations than did low RFI steers. However, plasma insulin concentrations and the ratio of glucose to insulin did not differ between the high and low RFI steers. Plasma insulin values in this study were greater than those reported in the literature (Yambayamba et al., 1996; Hersom et al., 2004), most likely due to the measurement of plasma insulin values with a bovine-specific insulin assay. The greater plasma glucose is a result of the greater feed intake of the high RFI steers; however, glucose metabolism does not seem to be altered because the ratios of glucose to insulin were similar between the high and low RFI steers. It seems that glucose metabolism or availability does not alter mitochondrial respiration rates.

	Footnotes

 
¹ This research was supported in part by a USDA special programs grant (No. 2004-34450-14578). The authors thank T. Elsasser (Growth Biology Laboratory, Animal and Natural Resources Institute, ARS, USDA, Beltsville, MD) for providing the guinea pig antibovine insulin antisera.

² Corresponding author: kerleym{at}missouri.edu

Received for publication May 18, 2005. Accepted for publication November 17, 2005.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 


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Deleo, D. 1994. Structure and function of the insulin receptor: Its role during lactation and fetal development. Ph.D. Diss., Curtin University of Technology, Bentley, Australia.

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Richardson, E. C., R. M. Herd, V. H. Oddy, J. M. Thompson, J. A. Archer, and P. F. Arthur. 2001. Body composition and implications for heat production of Angus steer progeny of parents selected for or against residual feed intake. Aust. J. Exp. Agric. 41:1065–1072.

Sodoyez, J. C., F. Sodoyez-Goffaux, M. M. Goff, A. E. Zimmerman, and E. R. Arquilla. 1975. [127-I]- or carrier-free [125-I]monoiodoinsulin J. Biol. Chem. 250:4268–4277.

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