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ANIMAL PRODUCTION |

,2

* Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, P. R. China; and
and
State Key Laboratory of Animal Nutrition, Beijing 100094, P. R. China
| Abstract |
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Key Words: carcass trait dietary manganese lipid oxidation meat quality manganese-containing superoxide dismutase activity manganese-containing superoxide dismutase mRNA level
| INTRODUCTION |
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In recent years, studies on the efficacies of organic trace elements in animals have received increasing attention. A study from our laboratory (Li et al., 2004
) showed that an organic Mn source with moderate chelation strength had a greater bioavailability than those with a weak or strong chelation strength or MnSO4·H2O, based on heart MnSOD mRNA levels in broilers. Apple et al. (2004)
found that chops from pigs fed diets containing 350 mg of Mn/kg from a Mn AA complex (Zinpro Corp., Eden Prairie, MN) had greater Japanese color scores and was darker than chops from pigs fed 350 mg of Mn/kg from MnSO4, but they did not determine MnSOD activity in the chops. Furthermore, no information is available regarding the effects of organic Mn sources on meat quality from broilers and modes of action.
Therefore, the objective of this study was to compare the efficacies of supplemental organic Mn sources with moderate chelation strength and MnSO4·H2O by investigating their effects on growth performance, carcass traits, quality and lipid oxidation of meat, relative enzyme activities in the abdominal fat and meat, and MnSOD gene expression in the meat of broilers.
| MATERIALS AND METHODS |
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In this study, birds were handled in accordance with guidelines (Yang and Diao, 1999
) approved by the Office of the Beijing Veterinarian. The experiment was conducted using a total of 336 commercial 1-d-old Arbor Acres male broilers. Birds were randomly allotted by BW to 1 of 7 treatments (8 replicate cages of 6 chicks per cage) in a completely randomized design involving a 2 x 3 factorial arrangement of treatments (2 levels of supplemental Mn x 3 sources of Mn, plus the control with no supplemental Mn). Broilers were housed in electrically heated, thermostatically controlled cages (100 x 50 x 45 cm) with fiberglass feeders and a 24-h constant light schedule.
The birds were allowed ad libitum access to the experimental diets and tap water containing 106 µg of Ca/ mL, 42 µg of Mg/mL, 0 µg of Cu/mL, 0 µg of Fe/mL, 0.01 µg of Mn/mL, and 3.3 µg of Zn/mL. Chicks were managed according to guidelines (Yang and Diao, 1999
) suggested by Arbor Acres Breeding Company in Beijing, China. Body weight, feed consumption, and incidence of leg abnormalities of each replicate cage were recorded at the end of each week. Incidence of leg abnormalities was calculated as a percentage of chicks within each cage with visual swelling at the articulatio tarsocruralis (tibiotarsal joint; Luo et al., 1991
).
The basal corn-soybean meal diets (Table 1
) were formulated to meet or exceed the requirements for starter and grower broilers (National Research Council, 1994
), except for Mn. Dietary treatments included the unsupplemented basal diet with Mn and supplemented with 100 or 200 mg of Mn/kg from MnSO4·H2O, Mn AA A with a chelation strength of 26.3 formation quotients (Qf; 8.34% Mn), or Mn AA B with a chelation strength of 45.3 Qf (6.48% Mn). All organic Mn sources used in the current study and their Qf values and Mn concentrations were determined by Li et al. (2004)
, and the organic Mn sources were obtained from independent distributors rather than directly from the product manufacturers.
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On d 42 of the experiment, 2 broilers from each cage were selected according to the average BW within the cage after a 12-h fast, weighed individually, killed by cutting the carotid arteries, and then were immediately bled. Heads and feet were removed from these birds, and then carcasses were eviscerated and weighed to determine the dressing percentage. Abdominal fat, breast, and leg muscle were removed and weighed to determine the content of abdominal fat, whole breast, and leg muscle. Some muscles from the breast and leg were stored individually in plastic bags at 80°C for testing crude fat content, Mn concentrations, and enzyme activities, whereas some muscles from the breast and leg were frozen in liquid N for assays of MnSOD gene expression and others were stored individually in plastic bags at 4°C for analyses of meat quality and MDA content. Some abdominal fat was also stored individually in plastic bags at 80°C for analysis of enzyme activities.
Meat Quality Measurements
Muscle pH. At 12 h after slaughter, the breast muscle and leg muscle pH was tested at a depth of 2.5 cm below the surface, using a pH meter (model pH 211, Hanna Instrument Inc., Villafranca Padovana, Italy) equipped with a spear electrode.
Color Measurements.
The Commission International de lEclairage color values for lightness (L*), redness (a*), and yellowness (b*; Commission International de lEclairage, 1976
) were determined for fresh breast and leg muscles using a chroma meter (model WSC-S, Shanghai Precision and Scientific Instrument Co., Shanghai, China) 5 min after slaughter.
Drip Loss Measurements.
Drip loss was measured as described by Remignon et al. (1996)
. The breast muscle (M. pectoralis profundus) and leg muscles (M. gastrocnemius and M. peroneus longus) were excised and weighed and then placed in a polyethylene bag and stored at 4°C. The muscle was removed from the bag 24 h postslaughter, wiped, and weighed to evaluate the drip loss, which was expressed as a percentage of the initial muscle weight.
Shear Force Measurements.
At 24 h after slaughter, the breast muscle and leg muscles were heated in plastic bags in a water bath at 96°C for 10 min. After cooling to room temperature, shear force was measured in triplicate, as described by Gwartney et al. (1992)
.
Intramuscular Fat Determination.
Crude fat content was determined in duplicate with 1.5-g samples of breast and leg muscles according to the Association of Official Analytical Chemists Method 960.39 (Soxhlet procedure; Association of Official Analytical Chemists, 1990
).
Measurement of MDA
At 24 h after slaughter, MDA content in breast or leg muscles was determined as described by Mak et al. (1983)
.
Mn Concentration
Manganese concentrations in Mn sources, diets, and muscles were determined by inductively coupled Ar plasma spectroscopy (Model 9000, Thermo Jarrell Ash, Waltham, MA), as described by Li et al. (2004)
. Approximately 0.2 g of each feed sample or 2 g of each muscle sample was weighed in triplicate and digested with 10 mL of HNO3 and 0.4 mL of HClO4 at 200°C in a 50-mL calibrated flask until the solution cleared, evaporated to almost dryness, and diluted 1:20 (feed samples; vol/ vol) or 1:10 (muscle samples; vol/vol) with 2% HNO3 before analyses. Validation of the mineral analysis was conducted using bovine liver powder [GBW (E) 080193, National Institute of Standards and Technology, Beijing, China] as a standard reference material.
Measurement of Enzyme Activities
The malate dehydrogenase (MDH) activity in abdominal fat was determined as described by Xu et al. (2003)
, and the activity of hormone-sensitive lipase (HSL) in abdominal fat was measured as described by Lu et al. (2006)
. The LPL activity in abdominal fat was determined according to methods described by Taskinen (1980)
. The MnSOD activity was measured by the nitrite method, as described by Li et al. (2004)
. The MnSOD activity in muscles was expressed as nitrite units per gram of fresh weight, and 1 nitrite unit was defined as the amount of enzyme needed to obtain 50% inhibition of nitrite formation.
Muscle RNA Extraction and MnSOD mRNA Analysis
Total RNA in breast and leg muscles was isolated using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturers instructions. The RNA concentration was estimated by measuring UV light absorbance at 260 nm (Ultrospec III, Perkin Elmer Cetus, Norwalk, CT). The MnSOD mRNA level was determined from samples using a semiquantitative reverse transcription PCR method as described by Li et al. (2004)
. ß-Actin was used as an internal control in all reactions. The MnSOD mRNA level was presented as the relative intensity ratio (arbitrary units) between the band intensity of MnSOD mRNA and ß-actin mRNA. Each PCR reaction was performed in duplicate on 2 individual preparations of reverse-transcribed cDNA.
Statistical Analysis
Data were analyzed by 2-way ANOVA using the GLM procedure (SAS Inst. Inc., Cary, NC). The model included the main effects of Mn source, supplemental Mn level, and their interaction. Cage was the experimental unit. An arcsine transformation was applied to the data on the incidence of leg abnormalities before statistical analysis. The linear and quadratic effects of the increasing levels of Mn were not determined because there were only 2 supplemental Mn levels in this study. Differences among means were tested by the LSD method, and P < 0.10 was considered to be statistically significant.
| RESULTS |
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Manganese source, supplemental Mn level, or the interaction between Mn source and level did not affect (P > 0.13) ADG, ADFI, or G:F during either experimental period (Tables 3
and 4
). Incidence of leg abnormality during the starter or grower phase was not influenced (P > 0.70) by Mn source or the interaction between Mn source and supplemental Mn level, but was influenced by Mn level (P < 0.05; Tables 3
and 4
). During the starter phase, chicks fed diets supplemented with 200 mg/kg of Mn from MnSO4·H2O, Mn AA A, or Mn AA B had lower (P < 0.10) incidences of leg abnormality than those fed the control diet or diets supplemented with 100 mg/kg of Mn. During the grower phase, chicks fed supplemental Mn had lower (P < 0.10) incidences of leg abnormality than those fed the control diet, and chicks fed diets supplemented with 200 mg of Mn/kg from MnSO4·H2O, Mn AA A, or Mn AA B had lower (P < 0.05) incidences of leg abnormality than those supplemented with 100 mg/kg of Mn.
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Differences among of Mn sources, supplemental Mn levels, or interactions between Mn source and supplemental Mn level were not detected (P > 0.21) in the dressing percentage and percentage of breast muscle or leg muscle (Table 5
). The percentage of abdominal fat was not affected (P > 0.84) by Mn source or the interaction between Mn source and supplemental Mn level, but was affected (P < 0.001; Table 5
) by supplemental Mn level. Birds fed supplemental Mn had lower (P < 0.10) percentages of abdominal fat than birds fed control diets. Birds fed supplemental Mn from organic sources had numerically lower percentages of abdominal fat that those fed supplemental Mn from MnSO4·H2O, but may not have been the result of treatment (P > 0.26).
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The Mn source, supplemental Mn level, or the interaction between Mn source and level did not affect (P > 0.10) pH, drip loss, shear force, i.m. fat, L* value or a* value in breast and leg muscle, or b* value in breast muscle (Tables 6
and 7
). Although b* value in leg muscle was not influenced (P > 0.95) by Mn source or the interaction between Mn source and level, it was influenced by Mn level (P < 0.001; Table 7
). Birds fed supplemental Mn at 200 mg/kg as 1 of 3 Mn sources had lower (P < 0.01) b* values in leg muscle than those fed the control diets or diets supplemented with 100 mg/kg of Mn.
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The Mn source, supplemental Mn level, or an interaction between Mn source and level did not affect (P > 0.11) MDA content in breast muscle (Table 6
). The MDA content in leg muscle was not affected by Mn source or the interaction between Mn source and supplemental Mn level, but was affected by supplemental Mn level (Table 7
). Birds fed diets supplemented with Mn had lower (P < 0.01) MDA contents in leg muscle than those fed the control diets.
LPL Activities in Abdominal Fat
The LPL activity in abdominal fat was not affected (P > 0.29) by the interaction between Mn source and supplemental Mn level, but was influenced (P < 0.0001) by supplemental Mn level, with birds fed supplemental Mn having lower (P < 0.05) LPL activities in abdominal fat than those fed the control diets (Table 8
). There was also an effect of Mn source (P < 0.05), with birds fed diets supplemented with Mn AA A or Mn AA B having lower (P < 0.05) LPL activities in abdominal fat than those fed diets supplemented with MnSO4·H2O.
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The MDH activity in abdominal fat was not affected (P > 0.29) by Mn source or the interaction between Mn source and supplemental Mn level, but was influenced (P < 0.10) by supplemental Mn level (Table 8
). Birds fed supplemental Mn had lower (P < 0.10) MDH activities in abdominal fat than those fed the control diets.
HSL in Abdominal Fat
The HSL activity in abdominal fat was not affected by (P > 0.42) Mn source and the interaction between Mn source and supplemental Mn level, but was influenced (P < 0.01) by supplemental Mn level (Table 8
). Birds fed supplemental Mn had greater (P < 0.05) HSL activities in abdominal fat than those fed the control diets. Birds fed supplemental Mn AA A and Mn AA B had numerically greater (P > 0.10) HSL activities in abdominal fat than those fed diets supplemented with MnSO4·H2O.
Mn Concentrations in Breast and Leg Muscles
The Mn source, supplemental Mn level, or the interaction between Mn source and supplemental Mn level did not affect (P > 0.22) Mn contents in breast and leg muscles (Table 9
). However, birds fed supplemental Mn as Mn AA A or Mn AA B had numerically greater Mn contents in breast and leg muscles.
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The MnSOD activity in breast or leg muscle was not affected (P > 0.40) by Mn source or the interaction between Mn source and supplemental Mn level, but was affected (P < 0.01) by supplemental Mn level (Table 9
). Birds fed diets supplemented with Mn had greater (P < 0.05) MnSOD activities in breast or leg muscle than those fed the control diets. Birds supplemented with Mn AA A or Mn AA B had numerically greater (P > 0.10) MnSOD activities in breast or leg muscle than those fed diets supplemented with MnSO4·H2O.
The MnSOD mRNA Levels in Breast and Leg Muscles
The interaction between Mn source and supplemental Mn level had no effect (P > 0.14) on MnSOD mRNA level in breast and leg muscle, and Mn source did not affect (P > 0.21) MnSOD mRNA level in breast muscle (Table 9
). However, MnSOD mRNA level in breast muscle was affected (P < 0.01) by supplemental Mn level, and MnSOD mRNA level in leg muscle was affected (P < 0.10) by Mn source and supplemental Mn level (Table 9
). Birds fed supplemental Mn had greater (P < 0.01) MnSOD mRNA levels in breast or leg muscle than those fed the control diets. Birds supplemented with Mn AA A had a greater (P < 0.05) MnSOD mRNA level in leg muscle than those supplemented with MnSO4·H2O.
| DISCUSSION |
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Results of the growth performance and incidence of leg abnormality of broilers from the current study are consistent with previous studies (Baker and Halpin, 1987
; Li et al., 2004
), indicating that growth performance and incidence of leg abnormality might be affected by factors other than Mn source and are not sensitive and specific indices for assessment of bioactivities of Mn sources for broilers. Manganese deficiency is one of the major factors inducing leg abnormalities of broilers. The lowest percentage of leg abnormality was observed at a supplemental Mn level of 200 mg/kg regardless of Mn source, suggesting that a much greater dietary Mn level might be required to minimize leg abnormality.
Sands and Smith (1999)
showed that addition of 240 mg/kg of Mn as Mn proteinate to broiler diets decreased abdominal fat deposition compared with an unsupplemented control (containing 19 mg of Mn/kg in the starter diet and 28 mg of Mn/kg in the grower diet). Lu et al. (2006)
also observed a decrease in abdominal fat percentage of broilers fed supplemental Mn as MnSO4·H2O. Results of the decreases in abdominal fat percentages in this study due to Mn additions agree with the aforementioned studies, indicating that Mn played an important role in reducing abdominal fat percentage of broilers.
Results from the meat quality evaluation in this study were consistent with a previous study (Lu et al., 2006
), but were not in agreement with observations of Roberts et al. (2001)
that loins from pigs fed 350 mg/ kg of Mn from a Mn amino complex (Mn AA; Zinpro Corp.) tended to be darker (lower L* values) than those from pigs fed the control diet or 350 mg of Mn/kg from MnSO4·H2O. This disagreement might be due to difference in animal species between the 2 studies. Furthermore, Roberts et al. (2002)
showed that leg muscle from pigs fed 350 mg/kg of Mn from Mn AA had greater Japanese and American color scores and tended to be less yellow than leg muscle from pigs fed the control or 700 mg/kg of Mn from Mn AA. Results from this study are consistent with the above study of Roberts et al. (2002)
and indicate that the addition of Mn to broiler diets decreased the b* value in leg muscle; however, the exact reason for these differences are unclear.
Lipid oxidation is a major cause of quality deterioration in meat and meat products and can give rise to rancidity and the formation of undesirable odors and flavors, which affect the functional, sensory, and nutritive values of meat products (Gray et al., 1996
). Malondialdehyde is a soluble degraded product of lipids and an indicater that can be widely used to reflect the extent of lipid oxidation in meat (Raharjo and Sofos, 1993
). Results from this study agree with previous findings (Roberts et al., 2002
) indicating that addition of Mn as either organic Mn sources or MnSO4·H2O to the Mn-deficient diet of broilers could reduce the level of oxidation in leg meat of broilers.
Luo et al. (1992)
reported that Mn concentration in breast muscle of broilers did not respond sensitively to dietary Mn. Li et al. (2004)
also found that the heart Mn of broilers was not a sensitive criterion for estimation of the bioavailability of supplemental sources. Results from the current study are consistent with the above findings, indicating that muscle Mn did not respond sensitively to dietary Mn source and level.
Lipoprotein lipase is the key enzyme that regulates adipose tissue triglyceride accumulation in broiler chickens (Mersmann, 1998
; Sato et al., 1999
). Yin et al. (2000)
reported that LPL activity increased as adipocyte number increased and could reflect the degree of adipocyte proliferation. Results from this study indicate that addition of Mn to broiler diets with no supplemental Mn could decrease LPL activity in abdominal adipose tissue, which agrees with results of Lu et al. (2006)
. Li et al. (2004)
showed that the Mn source with a moderate complex strength was the most available, and the Mn source with the strong complex strength had a tendency to be more available than the Mn source with the weak complex strength or reagent-grade MnSO4·H2O. In this study, Mn AA A and Mn AA B were both the Mn source with a moderate complex strength, and they were more effective than MnSO4·H2O in decreasing LPL activity in abdominal fat. The difference in bioactivity between organic and inorganic Mn sources might be due to 1 or both of the following reasons: 1) organic microelements could keep away from the interference of antinutritional factors in the gastrointestinal tract and be absorbed more effectively (Ashmead, 1993
) or 2) after organic microelements are absorbed, their metabolic pathway and mechanism might be different from those of inorganic microelements (Spears, 1989
; Swinkels et al., 1992 ). However, no direct experimental evidence for these hypotheses has been found. In the current study, the response of LPL activity to supplemental Mn source and level was similar to that of abdominal fat percentage, indicating that Mn could decrease abdominal lipid accumulation by decreasing LPL activity in abdominal adipose tissues. To our knowledge, whether Mn inhibited activity of LPL directly or indirectly is still unknown, and further experiments should be conducted to elucidate how Mn would affect LPL activity in abdominal fat.
Malate dehydrogenase is involved in synthesis of NADPH, and NADPH is an important factor in lipid synthesis (Shen et al., 1991
). Previous studies in broilers showed that hepatic activities of MDH were correlated with the proportion of abdominal fat (Whitehead et al., 1984
). In the current study, the response of MDH activity to dietary Mn source and level was similar to that of abdominal fat percentage, indicating that the change of abdominal fat deposition might be partially due to changes of MDH activity in abdominal fat. Furthermore, MDH activity in abdominal fat responded to dietary Mn less sensitively than LPL activity, suggesting that Mn might decrease abdominal fat deposition mainly by decreasing LPL activity in abdominal fat.
Hormone-sensitive lipase is an adipocyte enzyme that cleaves fatty acids from intracellular triacylglycerol (Mersmann, 1998
) and is considered to be the rate-limiting enzyme of lipolysis in the adipose tissue of animals (Fredrikson et al., 1981
; Belfrage et al., 1984
). The current results show that the response of HSL activity to dietary Mn source and level was opposite to that of abdominal fat percentage, indicating that the change of abdominal fat deposition might be partially due to changes of HSL activity in abdominal fat. However, further experiments are required to verify how Mn would affect HSL activity in abdominal fat.
Manganese-containing superoxide dismutase, which is located in the mitochondrial matrix, is the primary antioxidant enzyme that protects cells from oxidative stress by catalyzing dismutation of superoxide (O2°) to H2O2 and O2 in the mitochondria of eukaryotic cells (Flohe and Loschen, 1977
). When MnSOD activity decreases or is inhibited, membrane damage is observed as a consequence of peroxidative processes initiated by accumulation of free O2° (De Rosa et al., 1980
). Mice or chicks that are Mn-deficient have a low MnSOD activity in tissues (De Rosa et al., 1980
). Previous results from our laboratory indicate that broilers fed corn-soybean meal-based diets with no supplemental Mn had lower heart MnSOD activities than those fed diets supplemented with Mn (Luo et al., 1991
, 1992
; Li et al., 2004
) and had abnormal ultrastuctures of mitochondria and rough endoplasmic reticulum in heart cells (Luo et al., 1992
). Results from this study indicate that Mn sufficiency (the supplemental Mn level of 100 mg/kg) increased MnSOD activity in either breast or leg muscle, and Mn excess (the supplemental Mn level of 200 mg/kg) had no further effect on this parameter. In addition, MnSOD activity in leg muscle responded more sensitively than that in breast muscle, which was in agreement with previous results in this laboratory (Lu et al., 2006
). Results obtained from the current study also indicate that MnSOD activity in either breast or leg muscle lacked the sensitivity required to detect differences among Mn sources. In the current experiment, the addition of Mn increased MnSOD activity in breast muscle, but did not decrease MDA content in this tissue. These results indicate that factors other than MnSOD might be involved in lipid oxidation in breast meat. Furthermore, the current results showed that MnSOD activity in leg muscle increased, and MDA content in leg muscle decreased as a result of dietary Mn supplementation, suggesting that Mn might prevent lipid oxidation in leg muscle by increasing of MnSOD activity in it.
The mode of regulation in MnSOD gene expression in eukaryon is not clear, even though many investigations have focused on MnSOD gene structure and regulator sequences (Duttary et al., 1997
). Mice that are Mn-deficient have a low MnSOD mRNA in liver (Borrello et al., 1992
). Li et al. (2004)
reported that MnSOD mRNA levels in broiler heart increased linearly as dietary Mn levels increased, indicating that dietary Mn affected heart MnSOD gene transcription. In the current study, Mn sufficiency (the supplemental Mn level of 100 mg/ kg) increased MnSOD mRNA levels in either breast or leg muscle, and Mn excess (the supplemental Mn level of 200 mg/kg) had no further effect on this parameter. These results indicate that dietary Mn affected MnSOD gene transcription in either breast or leg muscle. Li et al. (2004)
showed that heart concentration of MnSOD mRNA was a more sensitive criterion than MnSOD activity in heart or other indices for estimation of bioavailability of organic Mn sources. Results from this study indicate that MnSOD mRNA level in leg muscle was a more sensitive criterion than MnSOD activity or other indices in breast or leg muscle for distinguishing the bioactivity of organic and inorganic Mn sources, and organic Mn with the moderate complex strength was more available than inorganic Mn in increasing the MnSOD mRNA level in leg muscle. Furthermore, the current results suggest that Mn might increase MnSOD activity in leg muscle by increasing the MnSOD mRNA level in it and then induce the decrease of MDA content in it.
| Footnotes |
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3 Current address: College of Animal Science and Technology, China Agricultural University, Beijing 100094, P. R. China. ![]()
2 Corresponding author: wlysz{at}263.net
Received for publication April 10, 2006. Accepted for publication September 29, 2006.
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