Livestock Science 135 (2011) 148152

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Livestock Science
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i

Effects of dietary delta-aminolevulinic acid and vitamin C on growth performance, immune organ weight and ferrum status in broiler chicks
J.P. Wang, L. Yan, J.H. Lee, T.X. Zhou, I.H. Kim
Department of Animal Resource & Science, Dankook University, No. 29 Anseodong, Cheonan, Choongnam 330-714, South Korea

a r t i c l e

i n f o

a b s t r a c t
A total of 480 one-day-old male broilers (Arbor Acre) were used to test the effects of dietary -aminolevulinic acid (ALA) and vitamin C (VC) supplementation on performance, blood characteristics, ferrum (Fe) status and immune organ weight. There were 6 treatments with 3 ALA (0, 5 and 10 kg 1) and 2 VC (0 and 500 mg kg 1) levels according to a 2 3 factorial arrangement. The experimental diets were fed to chicks for 5 weeks. Growth performance was not affected by ALA or VC supplementation in any experimental period. The interaction between ALA (10 mg kg 1) and VC signicantly increased the serum hemoglobin and Fe concentrations in serum, liver and breast meat (P b 0.05). Supplementation with ALA (10 mg kg 1) signicantly increased the RBC concentration at the end of the experiment (P b 0.05). The weights of liver, spleen, thymus and bursa of Fabricius were unaffected by dietary treatments. The effect of VC was shown to increase the L* value of breast meat in broilers. In conclusion, these results suggest that providing broilers with diets that contained ALA and VC can improve their Fe status without adversely affecting their growth performance, immune related blood proles and organ weights. 2010 Elsevier B.V. All rights reserved.

Article history: Received 16 August 2009 Received in revised form 30 June 2010 Accepted 30 June 2010 Keywords: -Aminolevulinic acid Vitamin C Fe status Immune response Broiler chicks

1. Introduction It is well known that Fe is an essential mineral that plays a critical role within cells that associate with oxygen utilization, enzymatic systems and overall cell function throughout the body. The absorption of Fe from the diet is thus determined more by meal composition than by the amount of Fe present in the diet. There are two forms of Fe found in the diet, non-heme Fe and heme Fe, with heme Fe being more efcient to be absorbed and utilized than non-heme Fe (Hallberg et al., 1989; Morris, 1987). Actually, approximately two-thirds of the Fe in the body is found in hemoglobin, while smaller amounts of Fe are found in myoglobin that helps supply oxygen to muscle and in enzymes that assist biochemical reactions in cells (Oates and

Corresponding author. Tel.: +82 41 550 3652; fax: +82 41 565 2949. E-mail address: inhokim@dankook.ac.kr (I.H. Kim). 1871-1413/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2010.06.161

West, 2006). The biosynthesis of heme initially occurs in the mitochondrion and involves the condensation of glycine and succinyl CoA to form -aminolevulinic acid (ALA). This reaction, catalyzed by ALA synthase, is the rate-limiting reaction of the heme biosynthesis process. This biosynthesis pathway is also controlled by the heme concentration, which exerts negative feedback on the activity of ALA synthase. Recently, it has been suggested that the addition of exogenous ALA may overcome the limitation of ALA synthase during heme synthesis, thereby causing more heme to be available in the body for utilization (Mateo et al., 2006; Wang et al., 2009). Based on the mechanism proposed above, several studies have been conducted to determine if ALA supplementation exerts benecial effects on livestock. The results of studies conducted in weanling pigs demonstrated that dietary 0.05% ALA supplementation could improve Fe, hemoglobin, and lymphocyte concentration (Min et al., 2004) and red blood cell counts (Mateo et al., 2006). Chen et al. (2008a) reported that 10 mg kg 1 ALA can improve DM, N digestibility, the levels of hemoglobin, and WBC counts (Chen et al., 2008a) of blood.

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However, the optimal levels of ALA being supplemented in other animals such as poultry are also of interest. The result of a previous study indicated that feeding ALA (5, 10, and 15 mg kg 1) to broilers exerted few effects in broilers (Chen et al., 2008b). In addition, the key role of ascorbic acid (vitamin C) for the absorption and utilization of dietary non-heme Fe is generally accepted (Hallberg et al., 1989; Hartiti et al., 1995). However, no study has been conducted so far to determine whether vitamin C (VC) can exert a positive effect on utilization of heme-iron (ALA). Therefore in the current study, we evaluate the effects of combined treatment with ALA and VC on performance, immune organ weight and Fe status in broilers. 2. Materials and methods 2.1. Experimental design and broiler husbandry All procedures used in this study were approved by the Animal Care and Use Committee of Dankook University. A total of 480 one-day-old male Arbor Acre broilers were obtained from a commercial hatchery (Yang Ji Company, Cheonan, Choongnam, Korea). The broilers were weighed and randomly placed into 24 battery cages (1 m 1 m) with 20 broilers in each cage, giving 4 replicate cages per dietary treatment. The average initial body weight was 43.0 5.0 g (SD) and this experiment was conducted for 35 days. The experimental design consisted of 6 dietary treatments resulting from 3 ALA addition levels (0, 5 and 10 mg kg 1) and 2 VC levels (0 and 500 mg kg 1). A 2-phase feeding program was used: a starter diet from 1 to 14 days and a nisher diet from 15 to 35 days. All broilers were fed maize-soybean meal-based diets that were

formulated to meet or exceed the National Research Council (1994) nutrient recommendations (Table 1). The ALA (EASY BIO System, Inc., Korea) was produced by recombinant Escherichia coli containing the Rhodobacter capsulatus hemA gene. The ALA was separated by HPLC to supply a nal product with a purity of 90%. The VC was supplied by Yuhan Co., Ltd (Korea) with the concentration over 91%. All the additives were added in the feed for 6 batches respectively. Broilers were housed in stainless steel battery cages with concrete oors covered with clean rice bran or hulls. All cages were in the same room and the temperature was kept at approximately 33 C during the rst 3 days and then reduced by 3 C every week until a temperature of 24 C was reached. Articial light was provided 24 h/day via uorescent lights. All diets were fed in mash form and feed and water were provided ad libitum throughout the experimental period. 2.2. Sampling and measurements The body weight (BW) and feed consumption were measured per cage at the beginning of the experiment, at day 14, and at the end of the experiment. This information was then used to determine the body weight gain (BWG) and feed intake (FI) per cage as well as the feed conversion ratio (FCR). At the end of the experiment, 12 chicks (3 chicks/cage) were randomly selected from each treatment and blood samples were then collected from the wing vein using a sterilized syringe. After collection, half of the sample was transferred into either a vacuum (clot activator with gel) or K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) immediately and then stored in a refrigerator at 4 C until further analysis. The remainder of the sample was centrifuged at 3000 g for 15 min to separate the serum. The Fe and total Fe binding capacity (TIBC), hemoglobin, total protein and albumin concentrations in the serum were then determined using an automatic biochemistry analyzer (HITACHI 747, Hitachi, Tokyo, Japan). In addition, the white blood cell (WBC), red blood cell (RBC), lymphocyte and hematocrit (HCT) levels were analyzed using an automatic blood analyzer (ADVIA 120, Bayer, NY, USA). At day 35, 12 broilers (3 chicks/cage) were randomly selected from each treatment group and then were slaughtered by cervical dislocation and the liver, spleen, thymus and bursa of Fabricius were then removed and cleaned from digesta. Organ weight was expressed as a percentage of BW. The breast meat Hunter L* (lightness), a* (redness), and b* (yellowness) values were measured using a Minolta CR410 chromameter (Konica Minolta Sensing Inc., Osaka, Japan). Duplicate pH values for each sample were measured using a pH meter (Fisher Scientic, Pittsburgh, PA). 2.3. Statistical analysis The data were analyzed by ANOVA as a 2 3 factorial arrangement of treatments, using a GLM procedure of SAS (SAS Institute, 1996). The cage served as the experimental unit during the feeding period, whereas individual bird was considered to be the experimental unit for other parameters. The nal model included main effects of ALA and VC, as well as the interaction between ALA and VC. When signicant (P b 0.05) interactions were observed, the means were

Table 1 Diet composition (as-fed basis). Ingredient, g kg 1 Maize Soybean meal (CP 48%) Maize gluten meal (CP 60%) Soybean oil Tricalcium phosphate Limestone Salt DL-methionine L-lysine-HCl Vitamin premix 1 Trace mineral premix 2 Analysis composition ME (MJ kg 1) Crude protein Lys Ca Available P Met + Cys Fe, mg kg 1 02 weeks (starter) 556.7 282.5 65.0 55.0 24.6 8.9 2.0 0.7 0.6 2.0 2.0 13.02 218.0 11.2 10.3 4.4 9.8 71 35 weeks (nisher) 632.1 246.1 35.0 48.9 22.9 7.5 2.0 0.7 0.8 2.0 2.0 12.82 189.0 10.4 9.1 3.1 9.8 68

1 Provided per kg of premix: 15,000 IU vitamin A, 3750 IU vitamin D3, 37.5 mg vitamin E, 2.55 mg vitamin K3, 3 mg vitamin B1, 7.5 mg vitamin B2, 4.5 mg vitamin B6, 24 g vitamin B12, 51 mg niacin, 1.5 mg folic acid, 126 mg biotin, and 13.5 mg pantothenic acid. 2 Provided per kg of diet: 37.5 mg Zn (as ZnSO4), 137.5 mg of Mn (MnO2), 37.5 mg of Fe (as FeSO47H2O), 3.75 mg of Cu (as CuSO45H2O), 0.83 mg of I (as KI), 0.23 mg of Se (as Na2SeO35H2O), and 1408 mg of choline.

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Table 2 Effects of -aminolevulinic acid (ALA) and vitamin C (VC) supplementation on growth performance in broilers. 1 VC, mg kg 1 ALA, mg kg 1 014 days BWG (g) FI (g) FCR 1535 days BWG (g) FI (g) FCR 035 days BWG (g) FI (g) FCR
1

0 0 360 467 1.30 1207 2230 1.85 1566 2697 1.72 5 368 476 1.29 1187 2257 1.90 1555 2732 1.76 10 360 465 1.29 1219 2250 1.85 1578 2714 1.72

500 0 357 461 1.29 1209 2245 1.86 1565 2706 1.73 5 361 474 1.31 1233 2292 1.86 1588 2766 1.74 10 381 478 1.26 1234 2342 1.90 1615 2819 1.75

SEM

P-value ALA VC 0.55 0.74 0.63 0.47 0.27 0.64 0.45 0.29 0.56 ALA VC 0.49 0.30 0.96 0.63 0.59 0.93 0.58 0.54 0.96

8 6 0.02 34 51 0.03 37 55 0.02

0.20 0.09 0.65 0.72 0.29 0.29 0.57 0.26 0.23

Each mean represents 4 cages with 20 chicks each per treatment.

compared based on the least signicant difference, and the variability was expressed as standard error of mean (SEM). 3. Results Weight gain, FI and FCR were not affected by diet in any period (Table 2). During the whole experiment, the mortality rate of the chicks shows no difference between these treatments (b 4%; data not shown). There was enhancement in Fe concentration in serum, liver and breast meat when ALA was added (Table 3, P b 0.05). The Fe concentration in liver and breast meat was higher (P b 0.05) in broilers that received VC (500 mg kg 1) supplemented diets than in those that received non-supplemented diets. The interaction between ALA (10 mg kg 1) and VC signicantly increased the serum hemoglobin and Fe concentrations in serum, liver and breast meat (P b 0.05). Supplementation with ALA (10 mg kg 1) signicantly increased the RBC concentration at the end of the experiment (P b 0.05, Table 4). The effect of VC was observed to increase (P b 0.05) the HCT levels. There was no difference in WBC, lymphocyte, albumin, and total protein concentrations among treatments. The weights of liver, spleen, thymus and bursa of Fabricius were unaffected by dietary treatments. The breast meat in broilers fed supplemental VC was lighter (P b 0.05) (L* value) than that of those from no VC addition treatments. The hunt color, redness (a*) and yellowness (b*) showed no difference among dietary treatments (Table 5).

4. Discussion Previous studies have shown that supplementation with ALA alone up to 50 mg kg 1 diet had no effect on the growth performance of weanling pigs (Mateo et al., 2006; Chen et al., 2008a) and broilers (Chen et al., 2008b). Even though this study evaluated the effects of ALA combined with VC supplementation, the results further conrmed that ALA supplementation did not inuence the growth performance of broilers. The response that the Fe depletion is associated with the tissue and growth performance is evident when the Fe-insufcient diet was used (Boling et al., 1998). The commercial diets utilized in the present study contains enough Fe concentration (71 and 68 mg kg 1 for starter and nisher feed) as recommended by NRC (1994), which is the same case in the previous studies. We found that broilers from ALA treatment had higher concentrations of Fe in the serum, liver and breast meat, but no difference was caused by ALA addition. Similarly, the results of these previous studies also demonstrated that serum Fe increased when the diets were supplemented with 10 mg kg 1 ALA in piglets (Chen et al., 2008a; Wang et al., 2009) and 5 mg kg 1 in laying hens (Chen et al., 2008c). ALA synthase, which is the reaction limiting enzyme during the formation of hemoglobin, is regulated by the production of heme via a feedback mechanism in most cells, with the exception of erythrocytes, in which it is regulated by the availability of Fe in the form of a Fesulfur cluster (Dring et al, 1998). It has also been reported that supplementation

Table 3 Effects of -aminolevulinic acid (ALA) and vitamin C (VC) supplementation on Fe status in broilers. 1 VC, mg kg 1 ALA, mg kg 1 Serum Hemoglobin (g/dL) Iron (g/dL) TIBC (g/dL) Liver (mg kg 1) Breast meat (mg kg 1)
a,b,c 1

0 0 8.65b 73.0b 167 68.3c 5.83c 5 8.97b 89.4ab 170 89.4b 6.08cb 10 8.89b 95.0ab 153 82.9b 6.21b

500 0 8.77b 65.6b 156 80.2b 6.09cb 5 9.26ab 94.6ab 161 93.5a 6.72b 10 9.47a 111.6a 196 100.9a 7.90a

SEM

P-value ALA VC 0.32 0.58 0.55 0.05 0.01 ALA VC 0.01 0.02 0.32 0.01 0.03

0.53 10.4 16 9.6 0.35

0.21 0.01 0.56 0.01 0.02

Means in the same row with different superscripts differ (P b 0.05). Each mean represents 4 cages with 12 chicks each per treatment.

J.P. Wang et al. / Livestock Science 135 (2011) 148152 Table 4 Effects of -aminolevulinic acid (ALA) and vitamin C (VC) supplementation on blood characteristics in broilers. 1 VC, mg kg 1 ALA, mg kg 1 RBC ( 106/mm3) WBC ( 104/mm3) Hematocrit (%) Lymphocyte (%) 2 Total protein (g/dL) Albumin (g/dL)
a,b 1

151

0 0 2.04b 31.8 24.2b 80.8 2.68 1.32 5 2.22a 35.8 25.1ab 84.0 3.08 1.50 10 2.21a 35.3 25.8ab 82.2 3.04 1.50

500 0 2.13ab 37.5 25.7ab 86.4 2.98 1.46 5 2.21a 36.5 26.4a 82.2 2.96 1.44 10 2.40a 38.4 26.7a 85.0 3.50 1.62

SEM

P-value ALA VC 0.19 0.08 0.03 0.35 0.95 0.33 ALA VC 0.98 0.29 0.74 0.31 0.28 0.44

0.06 2.10 0.70 2.80 0.24 0.08

0.02 0.30 0.09 0.92 0.15 0.09

Means in the same row with different superscripts differ (P b 0.05). Each mean represents 4 cages with 12 chicks each per treatment. 2 Values are presented as a percentage of total white blood cell count.

with exogenous ALA can bypass such feedback regulation, thereby enabling the production of additional heme (Mateo et al., 2006). However, Chen et al. (2008b) also previously reported that dietary ALA supplementation (5, 10, and 15 mg kg 1) had a marginal effect on serum Fe status in broilers. The discrepancy between these researches may due to variations in the animals, the ALA dosage, and also the Feresponse criteria. Underwood and Suttle (1999) indicated that the physiological inuences of Fe deciency are rstly Fe depletion in organs (such as liver, kidney and spleen), secondly in plasma Fe, hemoglobin, HCT, and myoglobin levels, and nally dysfunction and disease. Dietary Fe absorption is strongly affected by Fe status, the form consumed, and other constituents in the diet. Non-heme Fe is poorly absorbed compared with heme Fe and many nutritional factors are known to benet (vitamin C and meat) and inhibit (phytates, bers, polyphenols, and calcium) its absorption (Hallberg and Hulthen, 2000). Proteins such as transferrin, amino acids and VC can bind onto non-heme Fe ions and carry them across the walls of the duodenum and jejunum. Vitamin C is also able to reduce ferric Fe to ferrous Fe, thereby increasing its absorption by as much as 30% (Hallberg et al., 1989; Hallberg and Hulthen, 2000). Therefore, we evaluated broilers fed supplemental dietary ALA combined VC to determine if they performed better than those fed with ALA alone diets. As expected, the synergistic effect between VC and ALA was also observed to improve the hemoglobin and Fe concentrations in serum and tissue (liver and breast meat) in the current study. However, in spite of the increased Fe concentration in breast meat and the increased RBC levels in blood, there was

no change in the redness of the meat. Saddoris et al. (2003) reported that supplementation of the diet with Fe (Availa-Fe) resulted in a higher redness in the meat of pigs. Furthermore, Hagler et al. (1981) suggested that the increased redness of muscle may be due to accumulation of myoglobin in response to improved Fe status. However, Apple et al. (2000) suggested that minor improvements in pork color can be achieved by providing an additional 100 mg kg 1 of Fe, which is in agreement with results of the current study. On the other hand, the lighter color of the breast meat observed in treatment can be ascribed to a deoxidization trait of VC. Vitamin C, as an antioxidant, can prevent the myoglobin from oxidation to metmyoglobin, which has a brown color (Hui et al., 2001). It is well known that Fe plays an important role in immune development for both human and animals (Beard, 2001). Heme is considered to be the major functional form of Fe (Hallberg and Hulthen, 2000), and the ALA supplementation may increase the heme concentration, which may subsequently improve the iron utilization of Fe. The results of previous studies have suggested that dietary ALA supplementation (0.5% and 10 mg kg 1) has positive effects on the immunity of nursery pigs due to improvement of the body Fe status (Min et al., 2004; Chen et al., 2008a). However, in the current study, the ALA shown no effects on other blood characteristics (WBC, HCT and albumin) and weight of immune related organs. This is inconsistent with the result of Chen et al. (2008b), who reported that the ALA added at least 5 mg kg 1 increases WBC counts and relative immune organ weights (spleen and bursa of Fabricius) of broiler. The reason for this is not clear, and may be a high standard error

Table 5 Effects of -aminolevulinic acid (ALA) and vitamin C (VC) supplementation on organ weight and breast meat color in broilers. 1 VC, mg kg 1 ALA, mg kg 1 Organ weight (g/100 g BW) Liver Spleen Bursa of Fabricius Thymus Breast meat color L* a* b*
a,b 1

0 0 2.33 0.130 0.170 0.290 50.7b 11.8 12.3 5 2.38 0.122 0.173 0.285 53.0a 12.7 11.4 10 2.46 0.142 0.193 0.306 52.7a 12.9 10.3

500 0 2.34 0.139 0.182 0.357 53.8a 12.1 11.8 5 2.35 0.121 0.176 0.374 54.8a 12.2 10.8 10 2.74 0.141 0.170 0.366 54.7a 12.7 10.1

SEM

P-value ALA VC 0.49 0.38 0.98 0.07 0.01 0.78 0.55 ALA VC 0.56 0.97 0.45 0.94 0.50 0.59 0.97

0.42 0.08 0.07 0.03 0.92 0.74 0.93

0.16 0.89 0.88 0.68 0.07 0.27 0.08

Means in the same row with different superscripts differ (P b 0.05). Each mean represents 4 cages with 12 chicks each per treatment.

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