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Iron Glycine Chelate on Meat Color, Iron Status and Myoglobin Gene Regulation of M. longissimus dorsi in Weaning Pigs.

Byline: Zhao Zhuo, Shenglin Fang, Min Yue, Yizhen Wang and Jie Feng

Abstract

Effects of iron glycine chelate (Fe-Gly) on meat color, iron status and gene expression of myoglobin was studied in weaning pigs. A total of 180 pigs, weight about7.81 kg were selected and allotted to six dietary treatments. The treatments consisted of 0, 30, 60, 90, 120 mg Fe/kg diets from Fe-Glys, and 120 mg Fe/kg from FeSO4, respectively. Feeding trial lasted for 40 days, the results showed that a significant increase (P Less than 0.05) in a values for M. longissimus dorsi was observed when pigs were fed with Fe-Gly (60, 90 or 120 mg Fe/kg). A linear response on myoglobin in M. longissimus dorsi was observed with increasing Fe-Gly levels. In addition, the increasing Fe-Gly levels enhanced the content of total iron and heme iron in M. longissimus dorsi, but had no significant effects in nonheme iron contents between the treatments. Supplementation with 60 or 90 mg/kg Fe-Gly greatly increased the relative abundance of myoglobin mRNA (P Less than 0.05 or P Less than 0.01).

To conclude, adding Fe-Gly to diets for pigs improved the meat color, increased muscle total iron and heme iron content, and enhanced gene expression of myoglobin in M. longissimus dorsi pork. (c) 2013 Friends Science Publishers

Keywords: Iron glycine chelate; Meat color; Myoblogin; Weanling pigs

Introduction

Meat color is an important factor of meat products that influences consumer purchase decision and affects their perception of freshness of the product (Wilborn et al., 2004; Adzitey and Huda, 2012). Iron concentration has a direct effect on meat color (Lawrence et al., 2004), and red meat muscle contains higher heme iron and nonheme iron than light muscle (Kongkachuichai et al., 2002). Dietary Fe addition may increase concentration of muscle total iron and heme iron in pork (Yu et al., 2000). The primary pigment of myoglobin, a heme protein, is mainly responsible for meat color in animals (Mancini and Hunt, 2005). Previous studies showed that diets with addition of iron significantly increased the myoglobin content of the longissimus dorsi in calives (Bray et al., 1959) or in pork (Lin et al., 2002).

In studies with rats and humans, it was demonstrated that iron glycine chelate maintains high bioavailability. Our previous study also found that compared with ferrous sulfate, appropriate dietary Fe-Gly could improve growth performance, haematological characteristics in weanling pigs (Feng et al., 2007, 2009). Whereas data about the relationship between organic iron and meat color indexes in animal are limited. The objectives of our present research were to evaluate the effects of iron glycine chelate on meat color, iron status and myoglobin gene regulation of M. longissimus dorsi in weaning pigs.

Materials and Methods

Animals and Experimental Design

One hundred and eighty pigs (DurocxLandracexYorkshire), weighing about 7.81 kg were selected and allotted to six dietary treatments. Each treatment includes three replications with ten pigs per replicate.

The treatments consisted of 0 (control), 30, 60, 90, 120 mg Fe/kg diets from Fe-Gly, and 120 mg Fe/kg from FeSO4, respectively. The basal diet was formulated based on National Research Council (NRC, 1998) (Table 1). All pigs were given ad libitum access to feed and water in the 40 days feeding trial.

Sample Collection

On day 40, four pigs of each treatment were randomly selected and slaughtered. Chops of M. longissimus dorsi were removed for sensory, iron status and myoglobin analysis. For color measuring chops were stored at 4degC, other chops stored at -70degC until analysis.

Color Measurement

At 60 min after slaughtering, L (indicates lightness), a (indicates redness), and b (indicates yellowness) values were determined with a Minolta colorimeter (CR-310; Minolta, Tokokawa, Japan).

Measurement of Myoglobin

Measurement was performed as described by Henry and Bratzler (1960). Duplicate 25 g aliquots of the samples were minced in a blender for two minutes with 100 mL of cold distilled water. After blending, the solutions were centrifuged for 20 min at 2500 rpm. All centrifugation was carried out at temperatures of 4degC. Solid mono and dibasic potassium phosphate was added to precipitate the hemoglobin. To alleviate the difficulty encountered in dissolving the monobasic potassium phosphate it was first ground and sifted through a 250-micron sieve. In calculating the concentration of myoglobin per gram of fresh tissue, the extinction coefficient of 11.5, equivalent to that of cyanomethemoglobin and a molecular weight of 16,500 for myoglobin was assumed.

Total Iron, Heme and Nonheme Iron Analysis

Total iron concents were determined on wet ashed samples according to Helrich (1990) method. An atomic absorption spectrometer (AA6501, Shimadzu Corp., Kyoto, Japan) was used to sample absorbance at a wavelength of 248.3 nm Comparisons were made to a standard curve using 0, 1, 2, 5, and 10 mg/kg of iron. A method of Schricker et al. (1982) was carried out to determine nonheme iron concentrations of M. longissimus dorsi.

Total RNA Extraction and Reverse-Transcription-PCR

Assay (RT-PCR)

Total RNA of M. longissimus dorsi was extracted using Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA). Primer sequences of myoglobin and 18S rRNA housekeeping gene for RT-PCR were designed as shown in Table 2. Components of PCR assay mixture (50 mL) contained: 1 mL cDNA product, 5 mL 10xPCR reaction buffer, 1 mL sense primer (20 mM), 1 mL antisense primer (20 mM) and 0.5 mL Taq DNA polymerase, 3 mL MgCl2 (25 mM), 1 mL dNTPs mix.

PCR for myoglobin and 18S rRNA was done under the following thermal cycles: myoglobin at 94degC for 2 min, 29 cycles (94degC for 45 sec, 58degC for 45 sec, 72degC for 1 min), 72degC for 10 min; 18S rRNA at 94degC for 2 min, 29 cycles (94degC for 45 sec, 53degC for 45 sec, 72degC for 1 min), 72degC for 10 min.

Expression of Myoglobin mRNA in M. longissimus dorsi

Quantitative real-time PCR (Q-PCR) reactions were performed on the real-time PCR detection system (Bio-Rad, Hercules, CA, USA). SYBR Green I was used as the fluorescence reporter. Q-PCR was performed in duplicate in 25 mL reaction mixtures under the following protocol: 95degC for 30 sec, 40 cycles (95degC for 30 sec, 58degC for 30 sec) 95degC for 1 min, 55degC for 1 min, followed by a final stage of

Table 1: Ingredient and chemical composition of the basal diet on an as-fed basis

Ingredient###g/kg###Nutrienta

Corn###543.5 DE (MJ/kg)###14.38

Soybean meal###170###Crude protein (g/kg)###207.2

Extruded-soybean###100###Calcium (g/kg)###10.5

Whey###80###Phosphorus (g/kg)###7.6

Fish meal###60###Lysine (g/kg)###13.5

Wheat middling###10###Fe (g/kg)###78.55

Calcium hydrogen phosphate###10###Cu (g/kg)###58.13

Limestone###8###Zn (g/kg)###146.2

Soybean oil###5

Vitamin mineral premixb###10

Salt###2

Lysine###1.5

aDE based on calculated values, others were analyzed value bSupplied the following per kilogram of diet: vitamin A 15,000 IU; vitamin D2 3000 IU; vitamin E 30 IU; vitamin B2 5 IU; vitamin B1 3.0 mg; vitamin B12 0.025 mg; biotin 0.06 mg; pantothenic acid 20 mg; nicotinic acid 15 mg; Cu 50 mg; Zn 120 mg; Mn 60 mg; Se 0.67 mg; Co 1 mg

Table 2: Specific primers for the myoglobin and 18S rRNA genes

Gene###Accession###Primer###Oligonucleotide sequence###Length

###number###source###(bp)

Myoglobin M14433###Pig###5'-GGGGAAGGTGGAGGCTGATGTC-###335

###3' (sense primer)

###5'-GGAACCTGGATGATGGCTTCTGAGT-

###3' (antisense primer)

18S###AY265350###Pig###5' - CTCCACCAACTAAGAACGG- 3'###375

rRNA###(sense primer)

###5' - AAGACGGACCAGAGCGAAA- 3'

###(antisense primer)

95degC for 15 sec, 60degC for 15 sec, and 95degC for 15 sec. The linearity of the dissociation curve was analyzed using the iCycler iQ software 3.0, and the threshold cycle (Ct) was determined. Each sample was analyzed in duplicate and normalized to18S rRNA as the following equation: DCtGENE = CtGENE [?] Ct18S rRNA. The fold change can be estimated by the formula: 2( ), where DDCtGENE = DCtGENE of the control [?] DCtGENE of each pig (Liao et al., 2007).

Data Analysis

General Line Model procedures of SAS were used for data analysis. The planned single-d.f. tests included the linear and quadratic effects of Fe-Gly, the control versus FeSO4 (120 mg Fe/kg), FeSO4 versus Fe-Gly treatments (120 mg Fe/kg). The relative abundance of mRNA for myoglobin after different treatments was compared on the basis of the myoglobin to 18S rRNA ratios. P Less than 0.05 was set as differences significant.

Results and Discussion

Meat Color

Compared with the control, Fe-Gly supplementation in diets (60, 90, or 120 mg Fe/kg) increased a values for M. longissimus dorsi (P Less than 0.05), and 120 mg Fe/kg as ferrous sulfate also improved a value (P Less than 0.05) (Table 3).

It was reported that a values in veal enhanced when calves were fed increasing levels of Fe (Gariepy et al.,1998). The red sensory of meat was positively correlated to a values (P Less than 0.01). Ludeen et al. (2004) found that increasing level of dietary organic iron improved the meat color of M. longissimus dorsi.

Myoblobin Concentrations

A linear response on myoglobin in M. longissimus dorsi was observed with increasing Fe-Gly levels (Fig. 1). There was no difference on myoblobin concentrations when diets were supplemented with Fe-Gly or FeSO4. Meat color is affected by the concentration and properties of the meat pigments' myoglobin (Olsson and Pickova, 2005). The results from the current experiment were consistent with the studies of Bray et al. (1959) and Lin et al. (2002), supplementation of diet with iron had a significant effect upon muscle myoglobin concentration in the longissimus dorsi muscle. Furthermore, the increased a value or meat color may be a result of enhanced myoglobin contents in response to dietary Fe level (Yu et al., 2000; Ludeen, 2004).

Iron Status

Iron concentration has a direct effect on meat color (Lawrence et al., 2004) and the red meat muscle contains higher heme and non-heme iron than light muscle (Kongkachuichai et al., 2002). Present study found that the total iron and heme iron contents increased when pigs were fed increasing levels of Fe-Gly (Table 4). Yu et al. (2000) reported that increasing iron concentration in the muscle agreed with the trend toward redness of skin color, and both total-iron and heme-iron concentrations increased significantly along with the level of Availa-Fe (0-120 mg/kg) supplement. However, it should be noted that supplementing swine diets with 50-150 mg/kg of Fe (Availa-Fe, produced by Zinpro Corp.) did not alter concentrations of LM total, heme and non-heme iron. This point needs further study.

Development of Myoglobin Expression (RT-PCR and Q-PCR Assay)

Compared with the control, supplementation Fe-Gly (60, 90,120 mg Fe/kg) enhanced the relative abundance of myoglobin (P Less than 0.05; Fig. 2). When the piglets were fed with 60, 90, or 120 mg/kg Fe-Gly, myoglobin mRNA expression were higher than the piglets of the control group in Q-PCR assay (Fig. 3). In addtion, myoglobin mRNA expression of M. longissimus dorsi peaked when pig fed with 90 mg/kg Fe-Gly among all the treatments.

The 18S rRNA mRNA levels did not differ among treatments.

Myoglobin, an iron-binding protein, is the main pigment of red muscles, and its concentration in muscle is the most important factor responsible for meat color. Bray et al. (1959) found that the myoglobin concentration of the longissimus dorsi was nearly twice as high in calves fed iron Fig. 1: Effects of Fe-Gly on content of myoglobin in M.longissimus dorsi from weaning pigs. Treatments consisted of: 0-120 mg Fe/kg diet from Fe-Gly groups and positive control (120P, 120 mg Fe/kg diet from FeSO4).

Values are means +- SD, ""indicates significant difference compared with the control Fig. 2: Effect of Fe-Gly on the expression of myoglobin in the M. longissimus dorsi of weanling pigs (A) Electrophoresis results of reverse-transcription-PCR for myoglobin and 18S rRNA in M. longissimus dorsi. Lane 1: control group; lane 6: 120 mg/kg FeSO4 group; lane 2-5: 30, 60, 90, 120 mg/kg Fe-Gly group (B) The integrated optical density (IOD) ratio of each band of myoglobin and 18S rRNA for the relative group. Myoglobin gene expression was shown as myoglobin to 18S rRNA ratios supplemented diets as in control animals. Lin et al. (2002) showed that the concentration of myoglobin in muscle was greatly increased with dietary Fe level (0-400 mg/kg). Therefore, it would be expected that myoglobin expression is increasing as iron levels.

Lacking information on myoglobin expression and iron levels, Stephen and Robert (1983) reported that treatment with a variety of Fe() chelates had no effect on myoglobin expression but enhanced both iron accumulation and ferritin synthesis in L6 cells. The early observation found that inorganic iron promotes protein synthesis in reticulocytes by stimulation of heme synthesis (Grayzel et al., 1966), and has no

Table 3: Effects of different levels of iron glycine on meat color of M. longissimus dorsi in weanling pigs a

Table 4: Total iron, heme iron and non-heme iron in meat from weanling pigs a (mg/kg)

aValues are presented as means: n=4 for per treatment and data reported on a fresh basis

bFe source

cStandard error of the mean

dNon-orthogonal comparisons between the control vs. FeSO4 (120 mg Fe of per kg diet), and the FeSO4 (120 mg/kg) vs. Fe-Gly treatments (30~120 mg Fe of per kg diet). Linear and quadratic effects of increasing dietary Fe supplementation (0~120 mg/kg) as Fe-Gly eFe addition (mg/kg)

Fig. 3: Myoglobin mRNA expression in M. longissimus dorsi of weanling pigs

The extracted mRNA was analyzed by Q-PCR. PCR products were quantified for relative levels of mRNA using image by comparing Mb with 18S rRNA. Treatments consisted of: 0-120 mg Fe/kg diet from Fe-Gly groups and positive control (120P, 120 mg Fe/kg diet from FeSO4 group). Values are means+-SD, ""indicates significant difference compared with the control effect on either myoglobin or total protein accumulation (Bailey et al., 1990). The results of the present study indicated that Fe-Gly could promote myoglobin protein synthesis. This point warrants further research.

Conclusion

Adding Fe-Gly to weaning pigs diets could improve meat color, increase muscle total iron and heme iron content, and enhance gene expression of myoglobin in M. longissimus dorsi of pork.

Acknowledgements

This research was supported by National "973" Key Science Project (No. 2012CB124705), National Natural Science Foundation (No. 31272398), Ministry of Education of China NCET program (No. NCET-10-0727) and the Natural Science Foundation of Zhejiang province, China (No. R3110085).

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Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Science, Zhejiang University, Hangzhou, P.R. China, 310058

For correspondence: fengj@zju.edu.cn

To cite this paper: Zhuo, Z., S. Fang, M. Yue, Y. Wang and J. Feng, 2013. Iron glycine chelate on meat color, iron status and myoglobin gene regulation of M. longissimus dorsi in weaning pigs. Int. J. Agric. Biol., 15: 983-987
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Author:Zhuo, Zhao; Fang, Shenglin; Yue, Min; Wang, Yizhen; Feng, Jie
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9CHIN
Date:Oct 31, 2013
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