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ANIMAL NUTRITION |
* Department of Animal Sciences, University of Wisconsin, Madison 53706;
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Departamento de Ciencias Animales, Pontificia Universidad Católica de Chile, Santiago, Chile;
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Meat Science and Muscle Biology Lab, University of Wisconsin, Madison 53706; and and
Department of Kinesiology, University of Wisconsin, Madison 53706
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: growth • muscle • oxidation • rat • sorghum • tannin
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), and accumulation of lipid oxidation products adversely affects meat flavor (Faustman et al., 1998).
High tannin sorghums (HTS) contain proanthocyanidins (PA), flavonoid oligomers also known as condensed tannins (Krueger et al., 2003). Proanthocyanidins and other flavonoids have antioxidant activity in vitro. They inhibited low-density lipoprotein oxidation induced by Cu2+ (Porter et al., 2001) and inhibited oxidation of low-density lipoproteins mediated by a macrophage-like murine cell line (Masella et al., 2004). Natural flavonoids also retarded oxidation in processed meat products (Osada et al., 2000; Jenschke et al., 2004).
Effectiveness of flavonoids as antioxidants in vivo remains unclear (Halliwell et al. 2005) and few studies have tested their capacity to modulate oxidation in meats. Tang et al. (2001) showed that in frozen chicken meat, feeding 200 mg of catechins per kg of feed to the animals had equivalent antioxidant effect as feeding 200 mg of 
-tocopheryl acetate per kg of feed. Du et al. (2002) showed lower thiobarbituric acid-reactive substances (TBARS) in thigh meat and higher a* (redness) values in thigh patties after 7 d of storage at 4°C from chickens fed a diet containing 10% HTS.
Although HTS is considered of lower nutritive value than corn and has had detrimental effects on growth rate of rats, chicken, and cattle (Jambunathan and Mertz, 1973; Maxson et al., 1973; Nyamambi et al., 2000), Cousins et al. (1981) observed that diets with more than 75% HTS did not reduce ADG of growing pigs when compared with a corn-based diet. Furthermore, Stock et al. (1987) and Huck et al. (1998) noted that mixing corn and low-tannin sorghums had positive associative effects when finishing cattle. Thus, we hypothesize that a mixture of corn and HTS could reduce markers of oxidation on muscle of rats without having detrimental effects in growth.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). These diets contained HTS and corn at a ratio of 0:50, 20:30, 35:15, and 50:0 percent of the diet (S0, S20, S35, and S50, respectively; as-fed basis). The S0 diet was considered the control diet. Diets were manufactured by Harlan Teklad (Madison, WI) and are shown in Table 1. Diets were balanced to meet nutritional requirements of growing rats (NRC, 1995) and were provided as meal at approximately 0900 everyday. Feed and water were offered ad libitum. Diets were received 1 wk prior to the arrival of animals and stored refrigerated throughout the length of the experiment. A sample of each diet was collected once a week and stored at –80°C to prevent oxidation. High-tannin sorghum hybrid NK8830 was provided by Sorghum Partners Inc. (New Deal, TX) and contained 37.6 mg of tannins/g of grain, measured with the vanillin method of Price et al. (1978) using catechin (Aldrich Chemical Co., Milwaukee, WI) as a standard.
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This experiment was the first part of a larger study designed to study antioxidant effects of feeding HTS to meat-producing animals. Rats were selected because they have been widely used to study oxidative processes in skeletal muscle (Bejma and Ji, 1999; Hollander et al., 2000; Fu and Ji, 2003). Furthermore, availability of HTS in the United States is reduced, and by using rats the amount of grain and the time needed for the study were considerably reduced. Information gathered in this study will be used later to study oxidation in meat from larger species.
Two groups of 38 male Sprague Dawley rats each were purchased (Harlan, Indianapolis, IN). Rats were individually housed in wire-bottom stainless-steel cages, inside a room with constant temperature (22 ± 2°C) and a 12-h light cycle (light from 0700 to 1900). The first group was purchased at around 4 wk of age and the second group at around 12 wk of age. All rats arrived together and were fed the control diet for 1 wk to allow adaptation to the new conditions.
Animals within each age group were randomly allocated to 4 diets containing different concentrations of HTS in a completely randomized design. The younger group of rats was fed the experimental diets for 10 wk (10W), whereas the older group was fed the experimental diets for 2 wk (2W); therefore, rats were all killed at approximately 15 wk of age. Due to facility restrictions, groups 2W-S0, 2W-S50, 10W-S0, and 10W-S50 had 10 animals per group and groups 2W-S20, 2W-S35, 10W-S20, and 10W-S35 had 9 animals per group.
Body weight was measured at approximately 0900, without feed and water restriction. Average daily feed intake and BW were measured and ADG and G:F were calculated for d 1, 2, 3, 7, 10, and 14 in 2W groups and d 1, 2, 3, 7 and once weekly thereafter for group 10W. Average BW (± SD) of the rats at the beginning of the experiment was 329 ± 10.5 and 151 ± 9.0 g for the 2W and the 10W groups, respectively.
Tissue Sampling and Preparation
All rats were killed at 15 wk of age. Rats were individually moved to an adjacent room and killed by decapitation within 3 min of leaving the housing room. The lumbar area of the LM and the whole soleus muscle (SM) from one side of the carcass were sampled immediately postmortem. Muscles were selected based on their differences in oxidative metabolism. Whereas LM is a type IIB, fast-glycolytic muscle (Morales-Lopez et al., 1992), SM is a type I muscle, with a slow-oxidative metabolism (Hollander et al., 2000). A 3-cm piece of the central lobule of the liver was also removed. All tissue samples were frozen by submersion in liquid nitrogen and stored at –80°C. Carcasses were cooled at room temperature for 2 h and transferred to a refrigerator at 4°C. After 48 h of refrigerated storage, the LM and SM from the other side of the carcass were excised and stored wrapped in O2-permeable film (polyvinyl chloride wrap; Fisher Scientific, Pittsburg, PA) at 4°C in the dark. After aging for 6 d, the samples were stored at –80°C to prevent further oxidation.
A 10% (wt/vol) tissue homogenate was prepared in ice-cold 5 mM potassium phosphate buffer (pH 7.4) using a Potter-Elvehjem Teflon glass homogenizer (A. H. Thomas Co., Philadelphia, PA). The buffer also contained 0.1% (vol/vol) Triton X-100; leupeptin, 1 µg/mL; pepstatin, 1.4 µg/mL; and aprotinin, 1 µg/mL (Sigma, St. Louis, MO; Bejma and Ji, 1999). Feed homogenates were prepared by mixing feed samples and homogenization buffer at a 1:5 (wt/vol) ratio and were homogenized using a Polytron homogenizer (Kinematica AG, Lucerne, Switzerland) for 30 s at medium speed.
Protein Oxidation
Carbonyl content was used as a protein-oxidation marker in muscles and liver and was evaluated according to the method of Levine et al. (1990). The crude homogenate was centrifuged at 500 x g for 3 min. A 0.1-mL aliquot of supernatant was used to measure protein according to Bradford (1976) with BSA as the standard (Sigma). Homogenate supernatant (0.9 mL) was transferred into a microcentrifuge tube, 0.1 mL of 10% (wt/vol) streptomycin sulfate in 50 mM HEPES buffer was added, and the tube was vortexed vigorously. After a 15-min incubation at room temperature, the tube was centrifuged at 6,000 x g for 10 min at 4°C and 250 µL of supernatant was transferred into each of 3 microcentrifuge tubes. An equal volume (250 µL) of 10 mM dinitrophenylhydrazine prepared in 2 N HCl was added to 2 of the tubes, and the third tube received 250 µL of 2 N HCl to be used as a blank. Tubes were vigorously vortexed for 30 s and incubated for 1 h in the dark at room temperature, with periodic vortexing. Trichloroacetic acid (20% wt/vol; 330 µL) was added to each tube, vortexed, and incubated in ice for 10 min. After incubation, tubes were centrifuged at 14,000 x g for 10 min at 4°C, and the supernatant was carefully discarded. The pellet was washed with 1 mL of ethyl acetate:ethanol 1:1 (vol/vol) and centrifuged at 14,000 x g for 10 min at 4°C. The wash was repeated 2 more times. The pellet was finally dissolved in 1 mL of 6 M guanidine-HCl in 20 mM KH2PO4 (pH 2.3), and absorbance was read at 366 nm. The absorbance of the blank was discounted from the average absorbance of the duplicate samples, and carbonyl content was estimated using the molar absorption coefficient of 22,000 M–1·cm–1 and expressed as nanomoles of carbonyl per milligram of protein.
Lipid Oxidation
Lipid oxidation in muscles, liver, and feed was evaluated by TBARS using a modification of the method of Uchiyama and Mihara (1978) by Bejma and Ji (1999). Three milliliters of a solution of 1% (vol/vol) H3PO4 and 280 mM ferrous sulfate were added to an aliquot of 200 µL of crude homogenate in a plastic centrifuge tube with a screw cap. One mililiter of 0.6% (wt/vol) 2-thiobarbituric acid aqueous solution and 92.6 µL of 100 mg/mL butylated hydroxytoluene in ethanol were added to the tube and immediately vortexed vigorously. Tubes were incubated 15 min in a boiling water bath and then allowed to cool at room temperature. After cooling, 4 mL of n-butanol were added and tubes were vortexed for 15 s, after which tubes were centrifuged at 1,100 x g for 10 min at room temperature. Absorbance of the butanol fraction was read at 520 and 535 nm. Absorbance of the sample was calculated by subtracting the absorbance at 520 nm from the absorbance at 535 nm. A standard curve of 1,1,3,3-tetraethoxypropane (MDA) was used as reference (Sigma). Because of water loss in muscle samples during storage, muscle TBARS was expressed as nanomoles of MDA per milligram of protein in the crude-muscle homogenate. In liver and feed samples, TBARS were expressed as nanomoles of MDA per gram of wet material.
Statistical Analysis
Carbonyl content and TBARS data were analyzed by ANOVA, using the GLM procedure (SAS Inst. Inc., Cary, NC). Liver and each muscle were analyzed independently. Factors in the model for liver analysis were diet, feeding period, and their interaction. Factors in the model for muscles analysis were diet, feeding period, muscle storage time, and their interactions. Comparison between HTS diets and control were made when the ANOVA was significant (P < 0.05) for diet or a diet interaction. If the feeding period x muscle storage time interaction was significant, a test of simple effect for diet was performed (Winer, 1971) and the HTS diets were compared against the control if the test was significant. A Dunnett-Hsu adjustment for multiple comparisons was used to compare treatments against S0 (control) within feeding period for liver and within feeding period and muscle storage time for each muscle.
Changes over time in BW, ADG, ADFI, and G:F were analyzed as repeated measures using the MIXED procedure of SAS. Covariance structures were chosen based on the methodology described by Littell et al. (2000, 2002). Factors in the model for BW, ADG, ADFI, and G:F were diet, days, and their interaction. Feeding period was not included as factor in the model because the days that were evaluated in each age group were too different, which prevented the program from fitting the unstructured covariance. Thus, each feeding period was analyzed independently. Polynomial regressions were fitted between day and BW, day and ADG, day and ADFI, and day and G:F. Up to cubic terms were evaluated in the regression models. When the F-test for type I sums of squares was significant (P < 0.05) for a given regression term, that term and all the terms of lower degree were included in the final model. Least squares means were estimated for each diet at each time point from the fitted regressions. Differences between control and HTS diets for BW, ADG, ADFI, and G:F were estimated by differences of least squares means within days.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Growth and Intake
Figure 1 contains the growth and performance data from rats fed 2 wk. Between d 7 and 10, two rats in group 2W-S20 broke a tooth and lost BW. Data from those 2 rats were removed from the analysis only at d 10. No differences between any sorghum diet and the control were observed in BW, ADG, and G:F. There was also no difference between S0 and HTS diets for ADFI, except a trend for a reduction in S35 and S50 at d 1 (P = 0.091 and 0.087, respectively).
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. Group 10W-S35 had greater BW (P = 0.049) than 10W-S0 at d 70 and tended to be greater (P < 0.1) than 10W-S0 from d 21 to 63. Group 10W-S35 had greater ADFI (P < 0.05) than 10W-S0 at d 1, 2, 3 and 7 and tended to be greater (P < 0.1) than 10W-S0 at d 14 and 21. Similarly, 10W-S35 had higher gain (P < 0.05) than 10W-S0 at d 1, 2, 3, and 7 and tended to be greater (P = 0.07) than 10W-S0 at d 14. No differences in G:F were observed between control and any HTS diet in 10W group.
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Greater feed intake was observed by Cousins et al. (1981) in pigs consuming diets with more than 75% HTS when compared with pigs fed corn. They did not observe differences in ADG. Similarly, Maxson et al. (1973) reported that steers fed HTS had greater intake than steers fed corn but they also had decreased ADG. In both studies feed:gain ratio [the reciprocal of the efficiency of gain (G:F)] was greater for the HTS-fed animals compared with the corn-fed controls. We observed greater intakes and ADG only in animals fed the 35% HTS diet. Because G:F was not different, greater feed and energy intake seems to be the factor driving higher ADG. For the 50% HTS in the diet, astringency caused by PA could be reducing palatability to the point where rats reduced their intakes to amounts similar to the control. This curvilinear response seems consistent with observation of reduced ADG and feed intake in rats fed diets with more than 90% HTS (Jambunathan and Mertz, 1973; Mehansho et al., 1983). Similar to our results, Huck et al. (1998) observed that heifers fed a mixture of steam-flaked corn and steam-flaked sorghum at a 75:25 ratio had the numerically greatest ADFI and ADG compared with pure steam-flaked corn, pure steam-flaked sorghum, or mixtures of both at ratios 50:50 and 25:75, respectively. Considering that in general rats will consume food to meet their energy requirement (NRC, 1995), the reasons to explain why 10W-S35 group consumed more feed than the extra amount needed to compensate for the lower energy concentration of the diet are yet to be explored but could be related to the mixture of grains used.
To the best of our knowledge, this is the first report showing rats fed PA-rich diets exhibiting greater ADG than corn-fed matches. In previous research with rats where reductions in growth rate were observed, the amounts of PA in the diets were much greater than what we used in our study. Joslyn and Glick (1968) used 5% of the diet as purified grape seed and quebracho tannins; Schaffer et al. (1974) used 79% HTS containing more than 40 mg of catechin equivalents/g of grain; Jambunathan and Mertz (1973) used 95% HTS with up to 68.8 mg of catechin equivalents/g of grain. Many of these studies also provided diets that could be deficient in several amino acids based on today’s NRC (1995) standards. These deficiencies could have been exacerbated if tannins in the diets reduced total-tract nitrogen digestibility (Lizardo et al., 1995). The higher ADG observed in our experiment in rats eating HTS could be explained by a combination of at least 2 factors: the moderate amounts of HTS we used having positive effects on feed and energy intake, and newer and more accurate knowledge of protein requirements of rats.
Markers of Oxidation
No differences in markers of oxidation were observed between control and any HTS diet in liver (Table 2). No differences between HTS diets and control were observed for TBARS in fresh and aged LM, and in fresh and aged SM (Figure 3). Thiobarbituric acid-reactive substances in the diets are shown in Figure 4. A linear effect of time for TBARS was observed (P < 0.001). Slopes of the regressions lines were greater than zero (P < 0.001) for all diets, indicating an increase in TBARS over time. Slope of diet S20 was greater than slope of diet S0 (1.692 ± 0.152 vs. 0.724 ± 0.152, P < 0.001).
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). There was also a tendency for 10W-S20 to be lower than 10W-S0 (P = 0.054). Similarly, carbonyls were lower in 2W-S50 (P = 0.013) compared with 2W-S0 in aged SM and though 2W-S20 and 2W-S35 were not different than 2W-S0, there seems to be a dose-related reduction in carbonyl content with greater concentration of HTS in the diet for aged SM in 2W rats.
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; Masella et al., 2004). However, effectiveness of PA as antioxidants in vivo remains unclear. Even with high dietary intake of flavonoids, concentrations of their unconjugated forms and metabolites in plasma may be below levels needed to increase antioxidant activity (Halliwell et al. 2005).
Tang et al. (2001) observed decreased TBARS in thigh and breast meat of chicken fed tea catechins after 1 mo of storage at –20°C. Eid et al. (2003) showed that inclusion in the diet of a green tea extract reduced TBARS in breast and liver of chickens only when corticosterone was included in the diet. Several studies have shown increased markers of oxidation in animals under stress conditions (Chirase et al., 2004; 
ahin and Gümü
lü, 2004). Taking in consideration these observations, it seems likely that the antioxidant effects of dietary polyphenols are only evident after muscle has been subjected to an oxidative challenge such as stress conditions imposed on the animals or air exposure of the meat. However, we did not observed differences in TBARS in fresh and aged muscle between animals fed the control diet and animals fed HTS diets.
Muscle TBARS accumulate over time following a sigmoid-shaped curve (Liu et al., 1996). The best time to observe differences in oxidation products is in the central portion of the curves, after lag time has passed and before an asymptote is reached. Evaluating only fresh muscle and 1 storage time (6 d) could have masked any difference among diets. Furthermore, LM and SM are muscles with different oxidative metabolism (Morales-Lopez et al., 1992; Hollander et al., 2000), and in our study they showed different rates of lipid oxidation. Thus, optimal time for the evaluation of differences in TBARS in both muscles is likely to be different.
In our experiment, TBARS values after 6 d of display in LM were below 1 nmol of MDA/mg of protein and did not show a marked increase compared with values in fresh LM (Figure 3
). In contrast, TBARS in SM increased from values between 0.4 and 1.04 nmol of MDA/mg of protein in fresh muscle to values between 5.9 and 12.7 nmol of MDA/mg of protein after 6 d of refrigerated storage. Faustman and Cassens (1990) observed that in 6-d postmortem samples of beef, endogenous reductants are still acting and able to reduce metmyoglobin aerobically. It is possible that in the same way as in beef, 6 d of storage at 4°C is not enough to deplete endogenous reductants in LM of rats and that a detectable increase in TBARS may take longer.
Carbonyl content in fresh LM and SM was about 5-fold greater in animals fed 10 wk compared with animals fed 2 wk. Because all rats were killed at approximately the same age (15 wk), the increase in protein oxidation is not related to age but could be related to the time the animals were exposed to the experimental conditions. We did not have control on the diets that the rats were fed up to 1 wk before the experiment began, so there could be an antioxidant effect carried-over from the diets fed at the rearing facilities. Increase in protein oxidation could also be related to stress levels in the animals because stress treatments increased makers of oxidation (Chirase et al., 2004; ahin and Gümülü, 2004). Rats fed 2 wk were individually housed for only 3 wk, whereas rats fed 10 wk were individually housed for 11 wk before they were killed. Because rats are social animals that tend to live in colonies (NRC, 1995), isolation in metabolic cages could be a source of psychological stress that might be affecting markers of oxidation after several weeks. Tsunada et al. (2003) observed that oxidation products in the diet could affect oxidation markers in intestinal tissue. Thus, it is also possible that the greater oxidation products in the diets after 10 wk compared with 2 wk of feeding (Figure 4) could be affecting the oxidation status of the fresh muscles.
The antioxidant effect of dietary HTS was not consistent among HTS diets and was only effective in reducing markers of protein oxidation. Antioxidant effect was different depending on the muscle analyzed, with reductions in carbonyl content in LM only in 10W rats and reduction in carbonyl content in SM only in 2W rats.
The results of our study indicate that inclusion of moderate amounts of HTS in the diet increased feed intake and growth rate of young, fast-growing rats without changing the efficiency of gain. We also showed an antioxidant effect of dietary HTS on proteins of rats’ muscle. Further studies with large meat-producing animals in which increasing levels of HTS are added to the diet are required. If similar results are observed in animals such as swine or cattle, the use of high tannin sorghum as animal feed should be reassessed.
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Footnotes |
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2 Corresponding author: jdreed{at}wisc.edu
Received for publication December 21, 2006. Accepted for publication August 6, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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ahin, E., and S. Gümülü. 2004. Cold-stress-induced modulation of antioxidant defence: Role of stressed conditions in tissue injury followed by protein oxidation and lipid peroxidation. Int. J. Biometeorol. 48:165–171.[CrossRef][Medline]This article has been cited by other articles:
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  R. E. Larrain, D. M. Schaefer, S. C. Arp, J. R. Claus, and J. D. Reed Finishing steers with diets based on corn, high-tannin sorghum, or a mix of both: Feedlot performance, carcass characteristics, and beef sensory attributes J Anim Sci, June 1, 2009; 87(6): 2089 - 2095. [Abstract] [Full Text] [PDF]
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= S0, control diet, no high-tannin sorghum; 
= S20, 20% high-tannin sorghum diet; 
= S35, 35% high-tannin sorghum diet; and • = S50, 50% high-tannin sorghum diet. 
