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Effect of δ-aminolevulinic acid on growth performance, nutrient digestibility, blood parameters and the immune response of weanling pigs challenged with Escherichia coli lipopolysaccharide
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Livestock Science 114 (2008) 108 – 116 www.elsevier.com/locate/livsci
Effect of δ-aminolevulinic acid on growth performance, nutrient digestibility, blood parameters and the immune response of weanling pigs challenged with Escherichia coli lipopolysaccharide Y.J. Chen, I.H. Kim ⁎, J.H. Cho, B.J. Min, J.S. Yoo, Q. Wang Department of Animal Resource and Science, Dankook University, No. 29 Anseodong, Cheonan, Choognam 330-714, South Korea Received 20 February 2007; received in revised form 6 April 2007; accepted 14 April 2007
Abstract This study investigated the effects of dietary δ-aminolevulinic acid (ALA) on growth performance, nutrient digestibility, blood parameters and whether ALA improved the immune response of weanling pigs challenged with Escherichia coli lipopolysaccharide (LPS). Eighty pigs (body weight = 7.21 ± 0.51 kg) were allotted to four dietary treatments, with four pens per treatment and five pigs per pen. Basal diets were supplemented with 0, 5, 10, and 15 mg/kg ALA (as-fed basis) and fed for 35 days. At the end of the feeding period, 10 pigs were selected from both the 0- and 10-mg/kg ALA treatment groups; five were injected i.p. with LPS (50 μg/kg BW) and the other five pigs with an equivalent amount of sterile saline, resulting a 2 × 2 factorial arrangement. Blood sample and rectal temperature data were collected at 0, 2, 4 and 12 h after challenge. Growth performance was not affected by dietary treatments over the total experimental period. However, dry matter (DM) and nitrogen (N) digestibility was improved in the 15-mg/kg ALA treatment group at day 35 (P b 0.05). Serum hemoglobin (Hb) and iron levels were also increased, with the 10-mg/kg ALA treatment showing the highest concentration (P b 0.05). On day 35, red (RBC) and white blood cell (WBC) counts were elevated, with the 5- and 10-mg/kg ALA treatments having the highest counts (P b 0.05). During challenge, LPS injection elevated rectal temperature at 2 and 4 h postchallenge (P b 0.05). Plasma cortisol concentration was also increased by LPS injection at 2 and 4 h postchallenge and an ALA-alleviating effect was evident at 2 h postchallenge (P b 0.01). Concentration of plasma insulin-like growth factor-I (IGF-I) was increased in the ALA-supplemented treatments at 2 h postchallenge (P b 0.05). LPS injection increased plasma tumor necrosis factor-α (TNF-α) concentrations at 2, 4 and 12 h (P b 0.01), while an ALA-alleviating effect was observed at 2 and 4 h postchallenge (P b 0.05 and P b 0.10, respectively). Challenge with LPS decreased WBC counts at 2 and 4 h postchallenge (P b 0.01). At 12 h postchallenge, RBC, WBC and lymphocyte counts were affected by LPS challenge, while an ALA effect was only observed on WBC count (P b 0.05). In conclusion, dietary supplementation of ALA in weanling pigs can improve DM and N digestibilities, and iron status and have a beneficial effect on the immune response during inflammatory challenge. © 2007 Elsevier B.V. All rights reserved. Keywords: Delta-aminolevulinic acid; Immune response; Lipopolysaccharide; Weanling pigs
1. Introduction ⁎ Corresponding author. Tel.: +82 41 550 3652; fax: +82 41 565 2949. E-mail address: [email protected] (I.H. Kim). 1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.04.015
Delta-aminolevulinic acid (ALA) has been used in photodynamic therapy for several years. Recent studies
Y.J. Chen et al. / Livestock Science 114 (2008) 108–116
have reported that ALA has the potential as a biodegradable herbicide, insecticide and growth promoter for plants (Hotta et al., 1997; Nishikawa and Murooka, 2001). It is also reported that ALA, which is the precursor of heme, is synthesized by the condensation of glycine and succinyl-CoA with ALA synthetase as coenzyme. This reaction is the committed step of heme synthesis and rate-limiting for the pathway. After several steps of intermediate reactions, ALA is transformed to protoporphyrin IX. Subsequently, an iron atom is inserted into the porphyrin ring of protoporphyrin IX with the help of ferrochelatase and heme is finally formed. According to this reaction mechanism, a recent hypothesis suggest that dietary supplementation of ALA in livestock can affect the synthesis of heme and positively influence the iron or hemoglobin status of animals. Previous research with pigs has indicated that iron is the most common mineral deficiency (Rincker et al., 2004). It is also suggested that iron deficiency can impair the function of the immune system of young pigs (Brock, 1994; Beard, 2001). In addition, the health status of pigs is related to growth performance, as a compromised immune system will limit the production efficiency. Investigations into ALA may provide a new strategy for optimizing growth and/or health of pigs, especially nursery pigs, which have an immature immune system. In this study, the effect of dietary supplementation of ALA on growth performance, nutrients digestibility and blood characteristics was assessed in weanling pigs. A secondary objective was to evaluate whether dietary ALA supplementation could improve immune responses during a period of inflammatory challenge. 2. Materials and methods 2.1. Feeding period 2.1.1. Animals, housing, and treatments The experimental protocol was approved by the Animal Care and Use Committee of Dankook University. A total of 80 pigs ([Landrace × Yorkshire] × Duroc), with an average initial body weight (BW) of 7.21 ± 0.51 kg, were used in a 35-day feeding experiment. At the start of the experiment, pigs were selected by weight and randomly allotted to one of four dietary treatments in a completely randomized block design. There were four replicate pens per treatment with five pigs per pen. Experimental diets (Table 1) were formulated to contain graded levels of ALA at 0, 5, 10, and 15 mg/kg (as-fed basis). The composition of diets changed with phases (days 0–7, 8–21 and 22–35) to meet or exceed NRC
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Table 1 Compositions of experimental diets (as-fed basis) Ingredients (g/kg)
Phase 1 (day 0–7)
Phase 2 (day 8–21)
Phase 3 (day 22–35)
Expanded corn Expanded oat Biscuit meal Soybean meal Fermented soybean meal Fish meal Soy oil Lactose Whey Milk product a Lecithin Monocalcium phosphate Organic acid Glucose Sugar Plasma powder L-lysine–HCL DL-methinine L-threonine Zinc oxide Choline Cl Vitamin premix b Mineral premix c Limestone Salt
53.5 100.0 – 80.0 78.0 50.0 41.5 100.0 165.0 130.0 5.0 12.5 10.0 50.0 40.0 65.0 1.2 2.6 7.7 3.0 2.0 1.0 2.0 – –
346.2 – 50.0 200.0 82.0 40.0 48.0 60.0 100.0 20.0 – 10.0 8.0 – 20.0 – 2.5 1.5 0.8 3.0 1.0 1.0 2.0 2.0 2.0
448.0 – 90.0 296.5 – 25.0 30.0 – 62.5 20.0 – 6.0 6.0 – – – 1.6 1.4 – 3.0 1.0 1.0 2.0 3.0 3.0
Chemical composition d ME (MJ/kg) Crude protein (g/kg) Lysine (g/kg) Methionine (g/kg) Calcium (g/kg) Phosphorus (g/kg)
14.8 220.0 15.7 6.0 8.0 7.6
14.8 210.0 14.1 4.9 7.8 7.6
14.6 205.0 13.3 4.7 7.5 6.4
a
Mainly contains 210 g/kg fat and 220 g/kg protein. Provided per kg of complete diet : vitamin A, 11,025 IU; vitamin D3, 1,103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, D-pantothenic, 29 mg; choline, 166 mg; and vitamin B12, 33 μg. c Provided per kg of complete diet : Fe, 200 mg; Cu, 12 mg; Zn, 200 mg; Mn, 8 mg; I, 0.28 mg; Se, 0.15 mg. d Calculated values. b
(1998) nutrient recommendations, regardless of treatments. The ALA (EASY BIO System, Korea) was produced by recombinant Escherichia coli containing the Rhodobacter capsulatus hemA gene. Pigs were housed in an environmentally controlled nursery room with 12 h of artificial lighting per day. The initial room temperature was maintained at 30 °C and decreased by 1 °C each week of the experiment. Each pen was provided with a stainless steel feeder and one nipple waterer that allowed for ad libitum access to feed and water throughout the experiment. Before the start of experiment, pigs were acclimated for 2 days to the above-
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mentioned experimental facility, environment conditions and basal diet. 2.1.2. Experimental procedures, sampling and analysis Individual pig BW and pen feed disappearance were determined at the termination of each dietary phase and utilized in the determination of average daily gain (ADG), average daily feed intake (ADFI) and gain:feed ratio (G:F). During each dietary phase, chromium oxide (Cr2O3) was incorporated into the diets as an indigestible marker at the level of 2 g/kg from day 0 to 7, 14 to 21, and 28 to 35. On day 7, 21 and 35, fecal samples were collected from at least two pigs per pen by rectal massage. All the fresh fecal and feed samples were stored in a refrigerator at − 20 °C until analysis. Determination of dry matter (DM) and nitrogen (N) digestibility was performed according to AOAC (1995) procedures. Chromium was analyzed by UV absorption spectrophotometry (Shimadzu, UV-1201, Japan). Nitrogen was measured using a Kjeltec 2300 Analyzer (Foss Tecator AB, Hoeganaes, Sweden). At initiation of the experiment, two pigs were randomly chosen from each pen (n = 32) and blood samples were collected via jugular venipuncture. Same pigs were bled again on day 21 and 35. At each collection time, the blood samples were collected into both non-heparinized and K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) to obtain serum and whole blood, respectively. The white blood cell (WBC), red blood cell
(RBC) and lymphocyte counts were analyzed by an automatic blood analyzer (ADVIA 120, Bayer, NY, USA). Serum samples were subsequently centrifuged (2000 ×g) for 30 min at 4 °C. Serum hemoglobin (Hb), total protein, immunoglobin G (IgG), albumin, iron concentrations and total iron-binding capacity (TIBC) were determined in an automatic biochemistry analyzer (HITACHI 747, Japan). 2.2. Challenge period 2.2.1. General procedures After the feeding trial, 20 pigs were selected from the 0- and 10-mg/kg ALA-supplemented treatment groups (10 pigs per treatment and at least two pigs from each replicate pen) to conduct subsequent challenge trials. Pigs were kept individually during this period. The average BW of the 20 pigs was 18.96 ± 0.66 kg. Five pigs from the both the 0-or 10-mg/kg ALA treatments were challenged with E. coli lipopolysaccharide (LPS) and the other five pigs were administered sterile saline to serve as controls. This allocation lead to a 2 × 2 factorial arrangement, where the main effects were level of ALA (0 or 10 mg/kg) and LPS challenge (50 μg/kg of BW or without). No vaccines or antibiotics were administered to the pigs before or during the experimental period. LPS (E. coli: Serotype O55:B5; Sigma Chemicals, St. Louis, MO, USA; catalog L-6529) was injected i.p. (50 μg/kg of BW) at the start of the challenge trial. The LPS dosage was based on the results of previous studies
Table 2 Effects of δ-aminolevulinic acid supplementation on growth performance in weanling pigs a Items
Phase 1 (day 0–7) ADG (g) ADFI (g) G:F Phase 2 (day 8–21) ADG (g) ADFI (g) G:F Phase 3 (day 22–35) ADG (g) ADFI (g) G:F Overall (day 0–35) ADG (g) ADFI (g) G:F a b
Dietary ALA level b (mg/kg)
SEM
0
5
10
15
234 247 0.946
223 276 0.808
227 253 0.897
226 258 0.876
391 646 0.605
413 671 0.615
402 661 0.608
613 973 0.630
615 960 0.641
448 697 0.643
453 714 0.634
Each mean represents four pens with five pigs per pen. ALA = δ-aminolevulinic acid.
P-values Linear
Quadratic
Cubic
14 8 0.048
0.75 0.84 0.62
0.73 0.17 0.24
0.78 0.05 0.16
405 642 0.631
19 18 0.032
0.73 0.81 0.66
0.63 0.27 0.84
0.59 0.77 0.77
612 963 0.634
649 993 0.654
10 19 0.019
0.04 0.47 0.39
0.12 0.29 0.99
0.37 0.86 0.88
451 707 0.638
467 706 0.661
9 11 0.014
0.21 0.83 0.17
0.54 0.69 0.80
0.56 0.31 0.31
Y.J. Chen et al. / Livestock Science 114 (2008) 108–116
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Table 3 Effects of δ-aminolevulinic acid supplementation on DM and N digestibility in weanling pigs a Items
Day 7 DM N Day 21 DM N Day 35 DM N a b
Dietary ALA level b (mg/kg)
SEM
0
5
10
15
0.879 0.827
0.877 0.823
0.866 0.808
0.877 0.810
0.009 0.015
0.762 0.696
0.803 0.714
0.772 0.700
0.782 0.705
0.836 0.833
0.847 0.838
0.855 0.845
0.890 0.880
P-values Linear
Quadratic
Cubic
0.67 0.33
0.48 0.83
0.47 0.67
0.019 0.028
0.71 0.70
0.40 0.89
0.23 0.16
0.009 0.010
b0.01 b0.01
0.17 0.16
0.43 0.58
Each mean represents four observations per treatment. ALA = δ-aminolevulinic acid.
(Matteri et al., 1998; Wright et al., 2000). The LPS solution (1 mg/ml) was made by diluting LPS with sterile saline. The control pigs were injected with equal amounts of sterile saline solution. 2.2.2. Blood sampling and analysis The blood samples were collected at 0, 2, 4 and 12 h following LPS administration to obtain whole blood and
serum samples, using the same procedures mentioned in Section 2.1.2. Half of the whole blood samples were subsequently centrifuged for 10 min at 5000 ×g and the plasma harvested. Thereafter, plasma samples were frozen rapidly in liquid N2 and stored at −20 °C until analysis. Immediately after each blood collection, rectal temperature was also determined with a digital electronic thermometer.
Table 4 Effects of δ-aminolevulinic acid supplementation on blood characteristics in weanling pigs a Items
Dietary ALA level b (mg/kg) 0
Hb (g/dL) Day 0 Day 21 Day 35 Iron (μg/dL) Day 0 Day 21 Day 35 Albumin (g/dL) Day 0 Day 21 Day 35 RBC (106, no./mm3) Day 0 Day 21 Day 35 WBC (103, no./mm3) Day 0 Day 21 Day 35 Lymphocyte c (%) Day 0 Day 21 Day 35 a b c
5
SEM 10
15
P-values Linear
Quadratic
Cubic
10.4 9.8 10.3
10.2 9.6 9.7
11.1 11.7 11.1
11.3 10.3 10.3
0.6 0.5 0.4
0.17 0.07 0.42
0.71 0.16 0.91
0.49 0.01 0.03
66.0 102.4 64.2
50.6 87.0 68.6
49.8 126.0 92.0
61.2 80.0 85.4
13.9 10.6 7.7
0.81 0.56 0.03
0.35 0.18 0.49
0.97 0.01 0.18
3.70 3.24 3.64
3.76 3.42 3.36
3.96 3.56 3.48
4.02 3.38 3.54
0.11 0.11 0.16
0.04 0.27 0.80
0.99 0.12 0.30
0.58 0.57 0.52
5.42 5.81 6.73
5.23 6.03 6.39
5.60 6.69 7.12
5.70 6.07 6.47
0.22 0.17 0.26
0.23 0.09 0.97
0.51 0.03 0.57
0.40 0.04 0.05
8.4 21.6 16.5
8.9 17.9 20.1
10.3 20.7 15.3
10.3 20.4 17.4
1.2 3.9 1.3
0.23 0.82 0.75
0.84 0.56 0.59
0.67 0.55 0.02
47.8 62.5 65.4
56.0 66.6 56.8
52.0 64.0 62.6
44.4 67.2 66.4
5.3 3.4 4.8
0.56 0.36 0.69
0.16 0.76 0.22
0.72 0.38 0.46
Each mean represents eight observations per treatment. ALA = δ-aminolevulinic acid. Values are presented as percentage of total white blood cell count.
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Plasma cortisol concentration was analyzed in a commercially available Coat-a-count cortisol kit (Diagnostic Products Corp., Los Angeles, CA, USA). Plasma tumor necrosis factor-α (TNF-α) was analyzed by a commercially available ELISA kit (Sanquin, Amsterdam, The Netherlands). Plasma insulin-like growth factor-I (IGF-I) was determined using a commercially available kit (Catalog: DSL-5600; Diagnostic System Laboratories Inc. Kits, Webster, TX, USA). Serum haptoglobin was determined using a commercially available assay kit (Porcine C-Reactive Protein Assay and Haptoglobin Assay, Tri-Delta Diagnostics, Inc., Morris Plains, NJ, USA). 2.3. Statistical analysis Data from the feeding period were analyzed as a completely randomized block design using the GLM procedure of SAS (1996). In addition, orthogonal comparisons were conducted to measure the linear, quadratic and cubic effects for increasing dietary concentrations of supplementary ALA. The challenge experiment was a 2 × 2 factorial arrangement; therefore, data from the challenge period were analyzed as a randomized complete block design using the GLM procedure of SAS (1996). The main effects included dietary treatment and LPS administration. Because no interactions were observed for any of the response criteria between diet complexity and LPS challenge, the data are not presented in the results. The pen served as the experimental unit during the feeding period, whereas individual pigs served as the experimental unit in the challenge period. Variability of all the data was expressed as standard error of the mean (SEM) and the chosen level of significance was P b 0.05, with P b 0.10 being considered a tendency. 3. Results 3.1. Feeding period 3.1.1. Growth performance During phase 1 (day 0–7), the inclusion of graded levels of ALA in diets did not affect ADG or G:F, whereas a cubic effect was observed on ADFI (P = 0.05), with the 5-mg/kg ALA treatment having the highest value (Table 2). ADG, ADFI and G:F were not affected by increasing level of ALA in the diets during phase 2. Although a linear effect was observed on ADG during phase 3 (P = 0.04), this effect was not evident in the overall period (day 0–35). Average daily feed intake and G:F were not affected by dietary
treatments in either phase 3 or during the overall experimental period. 3.1.2. Nutrients digestibility On day 7 and 21 of the feeding period, there were no significant differences in DM and N digestibility among all treatment groups (Table 3). However, both DM and N digestibilities were improved by the inclusion of ALA, with the 15-mg/kg ALA treatment having the highest digestibility at the end of feeding period (day 35). 3.1.3. Blood characteristics At the beginning of the experiment (day 0), all tested blood characteristics had no significant differences among the treatments with the exception of serum albumin concentration, the values of which increased linearly with increasing ALA supplementation level (Table 4). On day 21, Hb concentration was higher in the 10-mg/kg ALA treatment group (linear: P = 0.07; cubic: P = 0.01).
Table 5 Effects of δ-aminolevulinic acid supplementation on inflammation response challenged with LPS in weanling pigs a Items
− LPS CON
b
+LPS ALA
Temperature (°C) 0h 39.7 39.7 2h 39.9 40.5 4h 40.0 40.0 12 h 39.8 39.9 Cortisol (μg/dL) 0h 2.00 2.40 2h 3.85 2.05 4h 4.08 3.85 12 h 5.10 4.66 IGF-I (ng/mL) 0h 481 578 2h 411 513 4h 465 491 12 h 333 404 TNF-α (pg/mL) 0h 174 178 2h 209 134 4h 178 180 12 h 147 190 Haptoglobin (mg/dL) 0h 1.25 2.38 2h 1.88 1.50 4h 1.75 2.23 12 h 2.05 2.70 a
b
CON
b
SEM ALA
b
P-values ALA effect
LPS effect
39.5 40.6 41.2 40.2
39.7 40.8 40.7 39.6
0.2 0.3 0.4 0.2
0.80 0.31 0.63 0.42
0.64 0.03 0.04 0.94
1.70 15.27 21.93 7.73
2.50 11.10 16.40 5.03
0.42 0.87 2.71 0.60
0.29 b0.01 0.27 0.07
0.99 b0.01 b0.01 0.16
473 423 421 304
520 484 427 316
49 31 31 46
0.16 0.04 0.17 0.92
0.53 0.88 0.12 0.08
202 2020 2049 1884
150 1995 1955 1792
36 25 27 95
0.49 0.04 0.09 0.67
0.99 b0.01 b0.01 b0.01
2.20 4.20 1.85 2.00
2.78 3.40 2.15 2.93
0.59 0.94 0.32 0.64
0.39 0.14 0.62 0.28
0.35 0.42 0.50 0.94
Twenty pigs with initial BWof 18.19 ± 0.66 kg. Each mean represents five observations per treatment. b Abbreviations: CON, basal diet; ALA, basal diet with δ-aminolevulinic acid 10 mg/kg.
Y.J. Chen et al. / Livestock Science 114 (2008) 108–116 Table 6 Effects of δ-aminolevulinic acid supplementation on blood cell counts challenged with LPS in weanling pigs a Items
− LPS CON
b
RBC (×106/mm3) 0h 6.74 2h 6.74 4h 6.37 12 h 6.40 WBC (×103/mm3) 0h 16.3 2h 19.4 4h 19.3 12 h 19.9 Lymphocyte c (%) 0h 81.6 2h 70.2 4h 70.8 12 h 72.4
+LPS ALA
b
CON
b
SEM ALA
b
P-values ALA effect
LPS effect
6.64 6.75 6.38 6.31
7.17 6.08 7.29 5.81
6.56 7.17 6.41 4.82
0.31 0.33 0.39 0.25
0.29 0.12 0.22 0.08
0.56 0.67 0.29 b0.01
17.7 20.2 21.1 22.5
27.6 8.6 5.8 22.3
20.7 5.5 3.1 29.7
2.4 1.6 1.1 1.5
0.26 0.43 0.67 0.02
0.01 b0.01 b0.01 0.04
86.0 73.0 73.8 73.0
86.2 83.8 72.0 47.5
84.4 90.5 73.0 39.3
4.1 4.7 5.1 4.3
0.68 0.28 0.86 0.58
0.80 b0.01 0.86 b0.01
a Twenty pigs with initial BWof 18.19 ± 0.66 kg. Each mean represents five observations per treatment. b Abbreviations: CON, basal diet; ALA, basal diet with δ-aminolevulinic acid 10 mg/kg. c Values are presented as percentage of total white blood cell count.
Similarly, a cubic effect (P = 0.03) was also observed on day 35. On day 21, ALA supplementation had a cubic effect on serum iron concentration (P = 0.01), with the highest concentration in the 10-mg/kg ALA group. Addition of ALA also increased iron concentration linearly at day 35 (P = 0.03). Neither serum total protein, IgG concentrations nor TIBC were affected by ALA supplementation during each dietary phase (data not shown). The RBC concentration was increased in the 10-mg/ kg ALA treatment on day 21 (quadratic: P = 0.03; cubic: P = 0.04). Red blood cell count also exhibited a cubic response to dietary treatments, again with the 10-mg/kg ALA diet showing the highest level (P = 0.05) on day 35. On the other hand, WBC counts showed no difference on day 21; however, a significant difference was observed on day 35, with the highest values in the 5-mg/ kg ALA treatment group, intermediate values in the 0and 15-mg/kg ALA treatments, and lowest in the 10mg/kg ALA group (cubic: P = 0.05). 3.2. Challenge period Challenge with LPS increased rectal temperature at 2 and 4 h postchallenge (P = 0.03, P = 0.04; Table 5). Similarly, plasma cortisol concentrations were also increased at 2 and 4 h after LPS administration (P b 0.01). ALA affected (P b 0.01) plasma cortisol concentrations at
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2 h postchallenge, with a tendency (P = 0.07) being observed at 12 h postchallenge. Pigs fed diets with ALA had a higher plasma IGF-I concentration (P = 0.04) compared to pigs fed diets without ALA at 2 h postchallenge. At 12 h after LPS administration, IGF-I levels tended to increase in LPS-treated pigs compared to nontreated pigs (P =0.08). Plasma TNF-α concentration was significant elevated at 2, 4 and 12 h postchallenge (P b 0.01). Pigs fed diets with ALA had lower plasma TNF-α levels (P =0.04) than pigs fed diets without ALA at 2 h postchallenge, with a decreased tendency (P =0.09) being observed at 4 h postchallenge. Serum haptoglobin concentration did not respond to either ALA supplementation or LPS challenge at 0–12 h postchallenge. Red blood cell count was reduced by LPS challenge, with a tendency in the ALA effect (P = 0.08) being observed at 12 h postchallenge (Table 6). The WBC count decreased with LPS challenge at 2 and 4 h postchallenge; however, it increased at 12 h postchallenge, with a ALA effect also being observed — the effect in the ALA-supplemented groups being greater than in treatments with no ALA (P = 0.02). Lymphocyte counts were greater in LPS challenged treatments than non-challenged treatment, 2 and 12 h after injection (P b 0.01); however, such an effect was not observed at 4 h postadministration. 4. Discussion 4.1. Growth performance and nutrients digestibility In this study, even though ADG was affected by diet included 15 mg/kg ALA in phase 3, such an effect was absent for the overall period. Limited studies have been concerned with ALA on growth performance. Min et al. (2004) reported that supplementation of ALA, together with antibiotics, had a positive effect on nursery pig's ADG during a 20-day feeding trial. However, the exact reason for this response was not clarified. A conflicting result was obtained by Mateo et al. (2006), who also conducted a nursery experiment and found no beneficial effect of ALA supplementation on growth performance. Our results are similar to the report of Mateo et al. (2006). It should be noted that the levels of ALA in this and two above-mentioned studies differed greatly at 1 and 2 and 0.5 g/kg, respectively, which may explain the variation in the results. In addition, Mateo et al. (2006) also stated that the experimental sanitary conditions are probably another reason of the lack of growth response. Average daily feed intake was also influenced by dietary treatments during day 0–7 in the current study. This effect was unexpected as no study has previously
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reported that ALA could affect or had any relationship with feed intake, including the above-mentioned reports. In fact, the results of ADFI were not affected by dietary treatments during most experimental periods (phase 2, phase 3 and overall period). The significant difference that was only observed in phase 1 was likely due to the relative high rate of diet wastage when pigs had only a relatively small amount of feed intake. Interestingly, supplementation of 15 mg/kg ALA showed higher DM and N digestibilities at the end of the experimental period, which might explain the linear increase in ADG, observed during phase 3. However, it could not be ascertained clearly why DM and N digestibilities were improved by supplementation of ALA. 4.2. Blood characteristics during feeding period Iron concentration is related to Hb concentration; therefore, the Hb concentration in blood is an indicator of iron status (Rincker et al., 2004). At the end of the experiment, it was found that serum Hb concentration had similar trend to serum iron concentration. Zimmerman (1980) suggested that an adequate level of Hb is 10 g/dL, while 8 g/dL or b7 g/dL are borderline anemia and anemia levels, respectively. In the current study, the values of serum Hb and iron concentration were highest in the 10mg/kg ALA-supplemented treatment, indicated that iron status was improved by ALA (10 mg/kg). However, it should be noted that even though serum Hb and iron concentrations differed significantly among treatments, the values were still within an adequate range (9.62– 11.72 g/dL) during the total feeding period. Hb is formed by heme and globin, and previous research has demonstrated that heme synthesis is affected by ALA production (Cable et al., 2000); therefore, it can be deduced that ALA is related to serum hemoglobin and iron status, which is incorporate in these reactions. Serum albumin concentration differed among the treatments on day 0; this effect was not anticipated and may be due to the individual diversity of pigs in the different treatments. 4.3. Challenge responses The inflammatory and acute phase responses after challenge with endotoxin materials, such as LPS, have been well characterized in pig models (Johnson, 1997; Warren et al., 1997; Wright et al., 2000). To our knowledge, this is the first evaluation of the effect of ALA on the immune response in weanling pigs following an immunological challenge with LPS. It is commonly believed that the inflammation response is mediated by increased production of proin-
flammatory cytokines (Webel et al., 1997; Wright et al., 2000; Bosi et al., 2004). During the challenge phase, plasma cortisol, TNF-α concentrations and rectal temperature were significantly elevated in LPS treatments. These factors indicate that the challenge stimulated the pig's defense system and, therefore, presented a typical inflammation response. As reported in previous studies, antibiotics may interact with ALA, so the current study used formulated diets without antibiotics. Despite this, the pigs maintained a relatively optimal growth performance during the total feeding period. This might be largely attributed to the sanitary experimental facilities. Several studies have already indicated that a relatively clean environment, which has less potential pathogens, can decrease cytokine production compared with less sanitary facilities (Roura et al., 1991; Cromwell, 2000). From our data, it was also found that the measured parameters (rectal temperature, cortisol, IGF-I, TNF-α and haptoglobin) were similar in control pigs administered saline. On the other hand, some parameters were dramatically influenced by LPS administration. For example, cortisol and TNF-α concentrations, and rectal temperature, were lower in pigs fed the ALA diets with LPS challenge. These results agree with expectations, which indicated that inflammatory responses were alleviated in ALAsupplemented treatments and such a positive effect was probably present under pathogen challenge rather than the sanitary environment. In contrast to a previous report, IGF-I concentration was not affected by LPS injection. Hevener et al. (1997) suggested that IGF-I concentration was suppressed for 96 h after LPS injection (5 μg/kg) to finishing pigs. Wright et al. (2000) also reported an abrupt decrease in growing pigs administered LPS (100 μg/kg). Even though no LPS effect was evident in our data, an improved effect of ALA was observed at 2 h postchallenge, which response may be attribute to the growth-immune axis in response to immunological stress (Fan et al., 1995; Wright et al., 2000). Challenge stress is attenuated in pigs fed the ALA diets, with a subsequent higher IGF-I secretion. Haptoglobin is believed to be one of the acute phase proteins, related to endotoxin challenge. However, the concentration was not affected by LPS administration in the current study. This result was consistent with that of Wright et al. (2000), who reported that no elevation in haptoglobin was observed after LPS injection. Baumann and Gauldie (1994) suggested that a single dosage of LPS might not be sufficient to significant stimulate haptoglobin production. Dritz et al. (1996) also reported that repeated injection of LPS can increase haptoglobin
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concentration. Therefore, the discrepancy in the IGF-I and haptoglobin concentrations between the current and other challenge studies may be mainly due to the LPS dosage, age of pigs, route of pathogen administration or strains of E. coli used in the various experiments. It was evident from this study that exogenous ALA supplementation influenced hemoglobin and iron status in weanling pigs. Numerous studies have reported the effect of iron in maintaining resistance to infectious diseases (De Sousa et al., 1991; Brock, 1994). Hershko (1996) suggested that iron is involved in the regulation of cytokine production via its influence on second-messenger systems. Indeed, Hallquist et al. (1992) reported that bactericidal activity of macrophages was attenuated during iron deficiency. Our results also showed that immunity-related blood cell (WBC or RBC) production was positively influenced in the ALA-supplemented treatments during both feeding and challenge periods. Similar results were also reported by Mateo et al. (2006), who suggested that the RBC counts can be increased by dietary ALA supplementation. Thus, the alleviated inflammatory response found in the current study could be ascribed to elevated iron availability (heme–iron) or in circulation, and blood cell production, with a subsequent, positive influence on the immune system of piglets. 5. Conclusion In conclusion, exogenous supplementation of δaminolevulinic acid in weanling pig diets resulted in elevation of iron status and specific immune system parameters. Results suggest that δ-aminolevulinic acid has potential as a immunity enhancer in the diet of weanling pigs. This study was conducted under normal conditions (without iron deficiency); therefore, further research is necessary to evaluate the effect of δaminolevulinic acid on iron-deficient nursery pigs and its efficacy under commercial farm conditions where a greater disease challenge is present. In addition, the exact mechanism of δ-aminolevulinic acid action needs further investigation. References AOAC, 1995. Official Method of Analysis, 16th Edition. Association of Official Analytical Chemists, Washington, DC. Baumann, H., Gauldie, J., 1994. The acute phase response. Immunol. Today 15, 74–80. Beard, J.L., 2001. Iron biology in immune function, muscle metabolism and neuronal functioning. J. Nutr. 131, 568s–580s. Bosi, P., Casini, L., Finamore, A., Cremokolini, C., Merialdi, G., Trevisi, P., Nobili, F., Mengheri, E., 2004. Spray-dried plasma improves growth performance and reduces inflammatory status of weaned pigs challenged
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with enterotoxigenic Escherichia coli K88. J. Anim. Sci. 82, 1764–1772. Brock, J.H., 1994. Iron in infection, immunity, inflammation and neoplasia. In: Brock, J.H., Halliday, J.W., Pippard, M.J., Powell, L.W. (Eds.), Iron Metabolism in Health and Disease. W.B. Saunders, London, pp. 354–389. Cable, E.E., Miller, T.G., Isom, H.C., 2000. Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms. Arch. Biochem. Biophys. 384, 280–295. Cromwell, G.L., 2000. Why and how antibiotics are used in swine production. In: Shook, L.B. (Ed.), The Proceedings of the Pork Industry Conference on Addressing Issues of Antibiotic Use in Livestock Production. Univ. Illinois, Urbana, pp. 7–27. De Sousa, M., Reimao, R., Porto, G., Grady, R.W., Hilgartner, M.W., Giardina, P., 1991. Iron and lymphocytes: reciprocal regulatory interactions. In: Albertini, A., Lenfant, C.L., Mannucci, P.M., Sixma, J.J. (Eds.), Biotechnology of Plasma Proteins. Curr. Stud. Hematol. Blood Transfus., vol. 58. Karger, Basel, pp. 171–177. Dritz, S.S., Owen, K.Q., Goodband, R.D., Nelssen, J.L., Tokach, M.D., Chengappa, M.M., Blecha, F., 1996. Influence of lipopolysaccharide-induced immune challenge and diet complexity on growth performance and acute-phase protein production in segregated earlyweaned pigs. J. Anim. Sci. 74, 1620–1628. Fan, J., Char, D., Bagby, G.J., Gelato, M.C., Lang, C.H., 1995. Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding proteins by tumor necrosis factor. Am. J. Physiol. 269, R1204–R1212. Hallquist, N.A., McNeil, L.K., Lockwood, J.F., Sherman, A.R., 1992. Maternal-iron-deficiency effects on peritoneal macrophage and peritoneal natural-killer-cell cytotoxicity in rat pups. Am. J. Clin. Nutr. 55, 741–746. Hershko, C., 1996. Iron and infection. Iron Nutr. Health Dis. 22, 231–238. Hevener, W., Almond, G.W., Armstrong, J.D., Richards, G., 1997. Effects of acute endotoxemia on serum somatotropin and insulinlike growth factor I concentrations in prepubertal gilts. Am. J. Vet. Res. 58, 1010–1013. Hotta, Y., Tanaka, T., Takaoka, H., Takeuchi, Y., Konnai, M., 1997. New physiological effects of δ-aminolevulinic acid in plants: the increase of photosynthesis, chlorophyll content, and plant growth. Biosci. Biotechnol. Biochem. 61, 2025–2028. Johnson, R.W., 1997. Inhibition of growth by pro-inflammatory cytokines: an integrated view. J. Anim. Sci. 75, 1244–1255. Mateo, R.D., Morrow, J.L., Dailey, J.W., Ji, F., Kim, S.W., 2006. Use of δ-aminolevulinic acid in swine diet: effect on growth performance, behavioral characteristics and hematological/immune status in nursery pigs. Asian-Aust. J. Anim. Sci. 19, 97–101. Matteri, R.L., Klir, J.J., Fink, B.N., Johnson, R.W., 1998. Neuroendocrine-immune interactions in the neonate. Domest. Anim. Endocrinol. 15, 397–407. Min, B.J., Hong, J.W., Kwon, O.S., Kang, D.K., Kim, I.H., 2004. Influence of dietary δ-aminolevulinic acid supplement on growth performance and hematological changes in weaned pigs. J. Korean Soc. Food Sci. Nutr. 33, 1606–1610. Nishikawa, S., Murooka, Y., 2001. δ-aminolevulinic acid: production by fermentation, and agricultural and biomedical application. Biotechnol. Genet. Eng. Rev. 18, 149–170. NRC, 1998. Nutrient Requirement of Pigs, 10th Edition. National Research Council, Academy Press, Washington, DC. Rincker, M.J., Hill, G.M., Link, J.E., Rowntree, J.E., 2004. Effects of dietary iron supplementation on growth performance, hematological status, and whole-body mineral concentrations of nursery pigs. J. Anim. Sci. 82, 3189–3197.
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Roura, E., Homedes, J., Klasing, K.C., 1991. Potential mechanism of action of antibiotics as growth promoters: modulation of immunologic stress. Poult. Sci. 70, 102. SAS, 1996. SAS User's Guide. Release 6.12 Edition. SAS Institute. Inc., Cary NC. USA. Warren, E.J., Finck, B.N., Arkins, S., Kelley, K.W., Scamurra, R.W., Murtaugh, M.P., Johnson, R.W., 1997. Coincidental changes in behavior and plasma cortisol in unrestrained pigs after intracerebroventricular injection of tumor necrosis factor-α. Endocrinology 138, 2365–2371. Webel, D.M., Finck, B.N., Baker, D.H., Johnson, R.W., 1997. Time course of increased plasma cytokines, cortisol, and urea nitrogen in
pigs following intraperitoneal injection of lipopolysaccharide. J. Anim. Sci. 75, 1514–1520. Wright, K.J., Balaji, R., Hill, C.M., Dritz, S.S., Knoppel, E.L., Minton, J.E., 2000. Integrated adrenal, somatotropic, and immune responses of growing pigs to treatment with lipopolysaccharide. J. Anim. Sci. 78, 1892–1899. Zimmerman, D.R., 1980. Iron in swine nutrition. National Feed Ingredient Association Literature Review on Iron in Animal and Poultry Nutrition. National Feed Ingredient Association, Des Moines, Iowa.
Livestock Science 114 (2008) 108 – 116 www.elsevier.com/locate/livsci
Effect of δ-aminolevulinic acid on growth performance, nutrient digestibility, blood parameters and the immune response of weanling pigs challenged with Escherichia coli lipopolysaccharide Y.J. Chen, I.H. Kim ⁎, J.H. Cho, B.J. Min, J.S. Yoo, Q. Wang Department of Animal Resource and Science, Dankook University, No. 29 Anseodong, Cheonan, Choognam 330-714, South Korea Received 20 February 2007; received in revised form 6 April 2007; accepted 14 April 2007
Abstract This study investigated the effects of dietary δ-aminolevulinic acid (ALA) on growth performance, nutrient digestibility, blood parameters and whether ALA improved the immune response of weanling pigs challenged with Escherichia coli lipopolysaccharide (LPS). Eighty pigs (body weight = 7.21 ± 0.51 kg) were allotted to four dietary treatments, with four pens per treatment and five pigs per pen. Basal diets were supplemented with 0, 5, 10, and 15 mg/kg ALA (as-fed basis) and fed for 35 days. At the end of the feeding period, 10 pigs were selected from both the 0- and 10-mg/kg ALA treatment groups; five were injected i.p. with LPS (50 μg/kg BW) and the other five pigs with an equivalent amount of sterile saline, resulting a 2 × 2 factorial arrangement. Blood sample and rectal temperature data were collected at 0, 2, 4 and 12 h after challenge. Growth performance was not affected by dietary treatments over the total experimental period. However, dry matter (DM) and nitrogen (N) digestibility was improved in the 15-mg/kg ALA treatment group at day 35 (P b 0.05). Serum hemoglobin (Hb) and iron levels were also increased, with the 10-mg/kg ALA treatment showing the highest concentration (P b 0.05). On day 35, red (RBC) and white blood cell (WBC) counts were elevated, with the 5- and 10-mg/kg ALA treatments having the highest counts (P b 0.05). During challenge, LPS injection elevated rectal temperature at 2 and 4 h postchallenge (P b 0.05). Plasma cortisol concentration was also increased by LPS injection at 2 and 4 h postchallenge and an ALA-alleviating effect was evident at 2 h postchallenge (P b 0.01). Concentration of plasma insulin-like growth factor-I (IGF-I) was increased in the ALA-supplemented treatments at 2 h postchallenge (P b 0.05). LPS injection increased plasma tumor necrosis factor-α (TNF-α) concentrations at 2, 4 and 12 h (P b 0.01), while an ALA-alleviating effect was observed at 2 and 4 h postchallenge (P b 0.05 and P b 0.10, respectively). Challenge with LPS decreased WBC counts at 2 and 4 h postchallenge (P b 0.01). At 12 h postchallenge, RBC, WBC and lymphocyte counts were affected by LPS challenge, while an ALA effect was only observed on WBC count (P b 0.05). In conclusion, dietary supplementation of ALA in weanling pigs can improve DM and N digestibilities, and iron status and have a beneficial effect on the immune response during inflammatory challenge. © 2007 Elsevier B.V. All rights reserved. Keywords: Delta-aminolevulinic acid; Immune response; Lipopolysaccharide; Weanling pigs
1. Introduction ⁎ Corresponding author. Tel.: +82 41 550 3652; fax: +82 41 565 2949. E-mail address: [email protected] (I.H. Kim). 1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.04.015
Delta-aminolevulinic acid (ALA) has been used in photodynamic therapy for several years. Recent studies
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have reported that ALA has the potential as a biodegradable herbicide, insecticide and growth promoter for plants (Hotta et al., 1997; Nishikawa and Murooka, 2001). It is also reported that ALA, which is the precursor of heme, is synthesized by the condensation of glycine and succinyl-CoA with ALA synthetase as coenzyme. This reaction is the committed step of heme synthesis and rate-limiting for the pathway. After several steps of intermediate reactions, ALA is transformed to protoporphyrin IX. Subsequently, an iron atom is inserted into the porphyrin ring of protoporphyrin IX with the help of ferrochelatase and heme is finally formed. According to this reaction mechanism, a recent hypothesis suggest that dietary supplementation of ALA in livestock can affect the synthesis of heme and positively influence the iron or hemoglobin status of animals. Previous research with pigs has indicated that iron is the most common mineral deficiency (Rincker et al., 2004). It is also suggested that iron deficiency can impair the function of the immune system of young pigs (Brock, 1994; Beard, 2001). In addition, the health status of pigs is related to growth performance, as a compromised immune system will limit the production efficiency. Investigations into ALA may provide a new strategy for optimizing growth and/or health of pigs, especially nursery pigs, which have an immature immune system. In this study, the effect of dietary supplementation of ALA on growth performance, nutrients digestibility and blood characteristics was assessed in weanling pigs. A secondary objective was to evaluate whether dietary ALA supplementation could improve immune responses during a period of inflammatory challenge. 2. Materials and methods 2.1. Feeding period 2.1.1. Animals, housing, and treatments The experimental protocol was approved by the Animal Care and Use Committee of Dankook University. A total of 80 pigs ([Landrace × Yorkshire] × Duroc), with an average initial body weight (BW) of 7.21 ± 0.51 kg, were used in a 35-day feeding experiment. At the start of the experiment, pigs were selected by weight and randomly allotted to one of four dietary treatments in a completely randomized block design. There were four replicate pens per treatment with five pigs per pen. Experimental diets (Table 1) were formulated to contain graded levels of ALA at 0, 5, 10, and 15 mg/kg (as-fed basis). The composition of diets changed with phases (days 0–7, 8–21 and 22–35) to meet or exceed NRC
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Table 1 Compositions of experimental diets (as-fed basis) Ingredients (g/kg)
Phase 1 (day 0–7)
Phase 2 (day 8–21)
Phase 3 (day 22–35)
Expanded corn Expanded oat Biscuit meal Soybean meal Fermented soybean meal Fish meal Soy oil Lactose Whey Milk product a Lecithin Monocalcium phosphate Organic acid Glucose Sugar Plasma powder L-lysine–HCL DL-methinine L-threonine Zinc oxide Choline Cl Vitamin premix b Mineral premix c Limestone Salt
53.5 100.0 – 80.0 78.0 50.0 41.5 100.0 165.0 130.0 5.0 12.5 10.0 50.0 40.0 65.0 1.2 2.6 7.7 3.0 2.0 1.0 2.0 – –
346.2 – 50.0 200.0 82.0 40.0 48.0 60.0 100.0 20.0 – 10.0 8.0 – 20.0 – 2.5 1.5 0.8 3.0 1.0 1.0 2.0 2.0 2.0
448.0 – 90.0 296.5 – 25.0 30.0 – 62.5 20.0 – 6.0 6.0 – – – 1.6 1.4 – 3.0 1.0 1.0 2.0 3.0 3.0
Chemical composition d ME (MJ/kg) Crude protein (g/kg) Lysine (g/kg) Methionine (g/kg) Calcium (g/kg) Phosphorus (g/kg)
14.8 220.0 15.7 6.0 8.0 7.6
14.8 210.0 14.1 4.9 7.8 7.6
14.6 205.0 13.3 4.7 7.5 6.4
a
Mainly contains 210 g/kg fat and 220 g/kg protein. Provided per kg of complete diet : vitamin A, 11,025 IU; vitamin D3, 1,103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, D-pantothenic, 29 mg; choline, 166 mg; and vitamin B12, 33 μg. c Provided per kg of complete diet : Fe, 200 mg; Cu, 12 mg; Zn, 200 mg; Mn, 8 mg; I, 0.28 mg; Se, 0.15 mg. d Calculated values. b
(1998) nutrient recommendations, regardless of treatments. The ALA (EASY BIO System, Korea) was produced by recombinant Escherichia coli containing the Rhodobacter capsulatus hemA gene. Pigs were housed in an environmentally controlled nursery room with 12 h of artificial lighting per day. The initial room temperature was maintained at 30 °C and decreased by 1 °C each week of the experiment. Each pen was provided with a stainless steel feeder and one nipple waterer that allowed for ad libitum access to feed and water throughout the experiment. Before the start of experiment, pigs were acclimated for 2 days to the above-
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mentioned experimental facility, environment conditions and basal diet. 2.1.2. Experimental procedures, sampling and analysis Individual pig BW and pen feed disappearance were determined at the termination of each dietary phase and utilized in the determination of average daily gain (ADG), average daily feed intake (ADFI) and gain:feed ratio (G:F). During each dietary phase, chromium oxide (Cr2O3) was incorporated into the diets as an indigestible marker at the level of 2 g/kg from day 0 to 7, 14 to 21, and 28 to 35. On day 7, 21 and 35, fecal samples were collected from at least two pigs per pen by rectal massage. All the fresh fecal and feed samples were stored in a refrigerator at − 20 °C until analysis. Determination of dry matter (DM) and nitrogen (N) digestibility was performed according to AOAC (1995) procedures. Chromium was analyzed by UV absorption spectrophotometry (Shimadzu, UV-1201, Japan). Nitrogen was measured using a Kjeltec 2300 Analyzer (Foss Tecator AB, Hoeganaes, Sweden). At initiation of the experiment, two pigs were randomly chosen from each pen (n = 32) and blood samples were collected via jugular venipuncture. Same pigs were bled again on day 21 and 35. At each collection time, the blood samples were collected into both non-heparinized and K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) to obtain serum and whole blood, respectively. The white blood cell (WBC), red blood cell
(RBC) and lymphocyte counts were analyzed by an automatic blood analyzer (ADVIA 120, Bayer, NY, USA). Serum samples were subsequently centrifuged (2000 ×g) for 30 min at 4 °C. Serum hemoglobin (Hb), total protein, immunoglobin G (IgG), albumin, iron concentrations and total iron-binding capacity (TIBC) were determined in an automatic biochemistry analyzer (HITACHI 747, Japan). 2.2. Challenge period 2.2.1. General procedures After the feeding trial, 20 pigs were selected from the 0- and 10-mg/kg ALA-supplemented treatment groups (10 pigs per treatment and at least two pigs from each replicate pen) to conduct subsequent challenge trials. Pigs were kept individually during this period. The average BW of the 20 pigs was 18.96 ± 0.66 kg. Five pigs from the both the 0-or 10-mg/kg ALA treatments were challenged with E. coli lipopolysaccharide (LPS) and the other five pigs were administered sterile saline to serve as controls. This allocation lead to a 2 × 2 factorial arrangement, where the main effects were level of ALA (0 or 10 mg/kg) and LPS challenge (50 μg/kg of BW or without). No vaccines or antibiotics were administered to the pigs before or during the experimental period. LPS (E. coli: Serotype O55:B5; Sigma Chemicals, St. Louis, MO, USA; catalog L-6529) was injected i.p. (50 μg/kg of BW) at the start of the challenge trial. The LPS dosage was based on the results of previous studies
Table 2 Effects of δ-aminolevulinic acid supplementation on growth performance in weanling pigs a Items
Phase 1 (day 0–7) ADG (g) ADFI (g) G:F Phase 2 (day 8–21) ADG (g) ADFI (g) G:F Phase 3 (day 22–35) ADG (g) ADFI (g) G:F Overall (day 0–35) ADG (g) ADFI (g) G:F a b
Dietary ALA level b (mg/kg)
SEM
0
5
10
15
234 247 0.946
223 276 0.808
227 253 0.897
226 258 0.876
391 646 0.605
413 671 0.615
402 661 0.608
613 973 0.630
615 960 0.641
448 697 0.643
453 714 0.634
Each mean represents four pens with five pigs per pen. ALA = δ-aminolevulinic acid.
P-values Linear
Quadratic
Cubic
14 8 0.048
0.75 0.84 0.62
0.73 0.17 0.24
0.78 0.05 0.16
405 642 0.631
19 18 0.032
0.73 0.81 0.66
0.63 0.27 0.84
0.59 0.77 0.77
612 963 0.634
649 993 0.654
10 19 0.019
0.04 0.47 0.39
0.12 0.29 0.99
0.37 0.86 0.88
451 707 0.638
467 706 0.661
9 11 0.014
0.21 0.83 0.17
0.54 0.69 0.80
0.56 0.31 0.31
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Table 3 Effects of δ-aminolevulinic acid supplementation on DM and N digestibility in weanling pigs a Items
Day 7 DM N Day 21 DM N Day 35 DM N a b
Dietary ALA level b (mg/kg)
SEM
0
5
10
15
0.879 0.827
0.877 0.823
0.866 0.808
0.877 0.810
0.009 0.015
0.762 0.696
0.803 0.714
0.772 0.700
0.782 0.705
0.836 0.833
0.847 0.838
0.855 0.845
0.890 0.880
P-values Linear
Quadratic
Cubic
0.67 0.33
0.48 0.83
0.47 0.67
0.019 0.028
0.71 0.70
0.40 0.89
0.23 0.16
0.009 0.010
b0.01 b0.01
0.17 0.16
0.43 0.58
Each mean represents four observations per treatment. ALA = δ-aminolevulinic acid.
(Matteri et al., 1998; Wright et al., 2000). The LPS solution (1 mg/ml) was made by diluting LPS with sterile saline. The control pigs were injected with equal amounts of sterile saline solution. 2.2.2. Blood sampling and analysis The blood samples were collected at 0, 2, 4 and 12 h following LPS administration to obtain whole blood and
serum samples, using the same procedures mentioned in Section 2.1.2. Half of the whole blood samples were subsequently centrifuged for 10 min at 5000 ×g and the plasma harvested. Thereafter, plasma samples were frozen rapidly in liquid N2 and stored at −20 °C until analysis. Immediately after each blood collection, rectal temperature was also determined with a digital electronic thermometer.
Table 4 Effects of δ-aminolevulinic acid supplementation on blood characteristics in weanling pigs a Items
Dietary ALA level b (mg/kg) 0
Hb (g/dL) Day 0 Day 21 Day 35 Iron (μg/dL) Day 0 Day 21 Day 35 Albumin (g/dL) Day 0 Day 21 Day 35 RBC (106, no./mm3) Day 0 Day 21 Day 35 WBC (103, no./mm3) Day 0 Day 21 Day 35 Lymphocyte c (%) Day 0 Day 21 Day 35 a b c
5
SEM 10
15
P-values Linear
Quadratic
Cubic
10.4 9.8 10.3
10.2 9.6 9.7
11.1 11.7 11.1
11.3 10.3 10.3
0.6 0.5 0.4
0.17 0.07 0.42
0.71 0.16 0.91
0.49 0.01 0.03
66.0 102.4 64.2
50.6 87.0 68.6
49.8 126.0 92.0
61.2 80.0 85.4
13.9 10.6 7.7
0.81 0.56 0.03
0.35 0.18 0.49
0.97 0.01 0.18
3.70 3.24 3.64
3.76 3.42 3.36
3.96 3.56 3.48
4.02 3.38 3.54
0.11 0.11 0.16
0.04 0.27 0.80
0.99 0.12 0.30
0.58 0.57 0.52
5.42 5.81 6.73
5.23 6.03 6.39
5.60 6.69 7.12
5.70 6.07 6.47
0.22 0.17 0.26
0.23 0.09 0.97
0.51 0.03 0.57
0.40 0.04 0.05
8.4 21.6 16.5
8.9 17.9 20.1
10.3 20.7 15.3
10.3 20.4 17.4
1.2 3.9 1.3
0.23 0.82 0.75
0.84 0.56 0.59
0.67 0.55 0.02
47.8 62.5 65.4
56.0 66.6 56.8
52.0 64.0 62.6
44.4 67.2 66.4
5.3 3.4 4.8
0.56 0.36 0.69
0.16 0.76 0.22
0.72 0.38 0.46
Each mean represents eight observations per treatment. ALA = δ-aminolevulinic acid. Values are presented as percentage of total white blood cell count.
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Plasma cortisol concentration was analyzed in a commercially available Coat-a-count cortisol kit (Diagnostic Products Corp., Los Angeles, CA, USA). Plasma tumor necrosis factor-α (TNF-α) was analyzed by a commercially available ELISA kit (Sanquin, Amsterdam, The Netherlands). Plasma insulin-like growth factor-I (IGF-I) was determined using a commercially available kit (Catalog: DSL-5600; Diagnostic System Laboratories Inc. Kits, Webster, TX, USA). Serum haptoglobin was determined using a commercially available assay kit (Porcine C-Reactive Protein Assay and Haptoglobin Assay, Tri-Delta Diagnostics, Inc., Morris Plains, NJ, USA). 2.3. Statistical analysis Data from the feeding period were analyzed as a completely randomized block design using the GLM procedure of SAS (1996). In addition, orthogonal comparisons were conducted to measure the linear, quadratic and cubic effects for increasing dietary concentrations of supplementary ALA. The challenge experiment was a 2 × 2 factorial arrangement; therefore, data from the challenge period were analyzed as a randomized complete block design using the GLM procedure of SAS (1996). The main effects included dietary treatment and LPS administration. Because no interactions were observed for any of the response criteria between diet complexity and LPS challenge, the data are not presented in the results. The pen served as the experimental unit during the feeding period, whereas individual pigs served as the experimental unit in the challenge period. Variability of all the data was expressed as standard error of the mean (SEM) and the chosen level of significance was P b 0.05, with P b 0.10 being considered a tendency. 3. Results 3.1. Feeding period 3.1.1. Growth performance During phase 1 (day 0–7), the inclusion of graded levels of ALA in diets did not affect ADG or G:F, whereas a cubic effect was observed on ADFI (P = 0.05), with the 5-mg/kg ALA treatment having the highest value (Table 2). ADG, ADFI and G:F were not affected by increasing level of ALA in the diets during phase 2. Although a linear effect was observed on ADG during phase 3 (P = 0.04), this effect was not evident in the overall period (day 0–35). Average daily feed intake and G:F were not affected by dietary
treatments in either phase 3 or during the overall experimental period. 3.1.2. Nutrients digestibility On day 7 and 21 of the feeding period, there were no significant differences in DM and N digestibility among all treatment groups (Table 3). However, both DM and N digestibilities were improved by the inclusion of ALA, with the 15-mg/kg ALA treatment having the highest digestibility at the end of feeding period (day 35). 3.1.3. Blood characteristics At the beginning of the experiment (day 0), all tested blood characteristics had no significant differences among the treatments with the exception of serum albumin concentration, the values of which increased linearly with increasing ALA supplementation level (Table 4). On day 21, Hb concentration was higher in the 10-mg/kg ALA treatment group (linear: P = 0.07; cubic: P = 0.01).
Table 5 Effects of δ-aminolevulinic acid supplementation on inflammation response challenged with LPS in weanling pigs a Items
− LPS CON
b
+LPS ALA
Temperature (°C) 0h 39.7 39.7 2h 39.9 40.5 4h 40.0 40.0 12 h 39.8 39.9 Cortisol (μg/dL) 0h 2.00 2.40 2h 3.85 2.05 4h 4.08 3.85 12 h 5.10 4.66 IGF-I (ng/mL) 0h 481 578 2h 411 513 4h 465 491 12 h 333 404 TNF-α (pg/mL) 0h 174 178 2h 209 134 4h 178 180 12 h 147 190 Haptoglobin (mg/dL) 0h 1.25 2.38 2h 1.88 1.50 4h 1.75 2.23 12 h 2.05 2.70 a
b
CON
b
SEM ALA
b
P-values ALA effect
LPS effect
39.5 40.6 41.2 40.2
39.7 40.8 40.7 39.6
0.2 0.3 0.4 0.2
0.80 0.31 0.63 0.42
0.64 0.03 0.04 0.94
1.70 15.27 21.93 7.73
2.50 11.10 16.40 5.03
0.42 0.87 2.71 0.60
0.29 b0.01 0.27 0.07
0.99 b0.01 b0.01 0.16
473 423 421 304
520 484 427 316
49 31 31 46
0.16 0.04 0.17 0.92
0.53 0.88 0.12 0.08
202 2020 2049 1884
150 1995 1955 1792
36 25 27 95
0.49 0.04 0.09 0.67
0.99 b0.01 b0.01 b0.01
2.20 4.20 1.85 2.00
2.78 3.40 2.15 2.93
0.59 0.94 0.32 0.64
0.39 0.14 0.62 0.28
0.35 0.42 0.50 0.94
Twenty pigs with initial BWof 18.19 ± 0.66 kg. Each mean represents five observations per treatment. b Abbreviations: CON, basal diet; ALA, basal diet with δ-aminolevulinic acid 10 mg/kg.
Y.J. Chen et al. / Livestock Science 114 (2008) 108–116 Table 6 Effects of δ-aminolevulinic acid supplementation on blood cell counts challenged with LPS in weanling pigs a Items
− LPS CON
b
RBC (×106/mm3) 0h 6.74 2h 6.74 4h 6.37 12 h 6.40 WBC (×103/mm3) 0h 16.3 2h 19.4 4h 19.3 12 h 19.9 Lymphocyte c (%) 0h 81.6 2h 70.2 4h 70.8 12 h 72.4
+LPS ALA
b
CON
b
SEM ALA
b
P-values ALA effect
LPS effect
6.64 6.75 6.38 6.31
7.17 6.08 7.29 5.81
6.56 7.17 6.41 4.82
0.31 0.33 0.39 0.25
0.29 0.12 0.22 0.08
0.56 0.67 0.29 b0.01
17.7 20.2 21.1 22.5
27.6 8.6 5.8 22.3
20.7 5.5 3.1 29.7
2.4 1.6 1.1 1.5
0.26 0.43 0.67 0.02
0.01 b0.01 b0.01 0.04
86.0 73.0 73.8 73.0
86.2 83.8 72.0 47.5
84.4 90.5 73.0 39.3
4.1 4.7 5.1 4.3
0.68 0.28 0.86 0.58
0.80 b0.01 0.86 b0.01
a Twenty pigs with initial BWof 18.19 ± 0.66 kg. Each mean represents five observations per treatment. b Abbreviations: CON, basal diet; ALA, basal diet with δ-aminolevulinic acid 10 mg/kg. c Values are presented as percentage of total white blood cell count.
Similarly, a cubic effect (P = 0.03) was also observed on day 35. On day 21, ALA supplementation had a cubic effect on serum iron concentration (P = 0.01), with the highest concentration in the 10-mg/kg ALA group. Addition of ALA also increased iron concentration linearly at day 35 (P = 0.03). Neither serum total protein, IgG concentrations nor TIBC were affected by ALA supplementation during each dietary phase (data not shown). The RBC concentration was increased in the 10-mg/ kg ALA treatment on day 21 (quadratic: P = 0.03; cubic: P = 0.04). Red blood cell count also exhibited a cubic response to dietary treatments, again with the 10-mg/kg ALA diet showing the highest level (P = 0.05) on day 35. On the other hand, WBC counts showed no difference on day 21; however, a significant difference was observed on day 35, with the highest values in the 5-mg/ kg ALA treatment group, intermediate values in the 0and 15-mg/kg ALA treatments, and lowest in the 10mg/kg ALA group (cubic: P = 0.05). 3.2. Challenge period Challenge with LPS increased rectal temperature at 2 and 4 h postchallenge (P = 0.03, P = 0.04; Table 5). Similarly, plasma cortisol concentrations were also increased at 2 and 4 h after LPS administration (P b 0.01). ALA affected (P b 0.01) plasma cortisol concentrations at
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2 h postchallenge, with a tendency (P = 0.07) being observed at 12 h postchallenge. Pigs fed diets with ALA had a higher plasma IGF-I concentration (P = 0.04) compared to pigs fed diets without ALA at 2 h postchallenge. At 12 h after LPS administration, IGF-I levels tended to increase in LPS-treated pigs compared to nontreated pigs (P =0.08). Plasma TNF-α concentration was significant elevated at 2, 4 and 12 h postchallenge (P b 0.01). Pigs fed diets with ALA had lower plasma TNF-α levels (P =0.04) than pigs fed diets without ALA at 2 h postchallenge, with a decreased tendency (P =0.09) being observed at 4 h postchallenge. Serum haptoglobin concentration did not respond to either ALA supplementation or LPS challenge at 0–12 h postchallenge. Red blood cell count was reduced by LPS challenge, with a tendency in the ALA effect (P = 0.08) being observed at 12 h postchallenge (Table 6). The WBC count decreased with LPS challenge at 2 and 4 h postchallenge; however, it increased at 12 h postchallenge, with a ALA effect also being observed — the effect in the ALA-supplemented groups being greater than in treatments with no ALA (P = 0.02). Lymphocyte counts were greater in LPS challenged treatments than non-challenged treatment, 2 and 12 h after injection (P b 0.01); however, such an effect was not observed at 4 h postadministration. 4. Discussion 4.1. Growth performance and nutrients digestibility In this study, even though ADG was affected by diet included 15 mg/kg ALA in phase 3, such an effect was absent for the overall period. Limited studies have been concerned with ALA on growth performance. Min et al. (2004) reported that supplementation of ALA, together with antibiotics, had a positive effect on nursery pig's ADG during a 20-day feeding trial. However, the exact reason for this response was not clarified. A conflicting result was obtained by Mateo et al. (2006), who also conducted a nursery experiment and found no beneficial effect of ALA supplementation on growth performance. Our results are similar to the report of Mateo et al. (2006). It should be noted that the levels of ALA in this and two above-mentioned studies differed greatly at 1 and 2 and 0.5 g/kg, respectively, which may explain the variation in the results. In addition, Mateo et al. (2006) also stated that the experimental sanitary conditions are probably another reason of the lack of growth response. Average daily feed intake was also influenced by dietary treatments during day 0–7 in the current study. This effect was unexpected as no study has previously
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reported that ALA could affect or had any relationship with feed intake, including the above-mentioned reports. In fact, the results of ADFI were not affected by dietary treatments during most experimental periods (phase 2, phase 3 and overall period). The significant difference that was only observed in phase 1 was likely due to the relative high rate of diet wastage when pigs had only a relatively small amount of feed intake. Interestingly, supplementation of 15 mg/kg ALA showed higher DM and N digestibilities at the end of the experimental period, which might explain the linear increase in ADG, observed during phase 3. However, it could not be ascertained clearly why DM and N digestibilities were improved by supplementation of ALA. 4.2. Blood characteristics during feeding period Iron concentration is related to Hb concentration; therefore, the Hb concentration in blood is an indicator of iron status (Rincker et al., 2004). At the end of the experiment, it was found that serum Hb concentration had similar trend to serum iron concentration. Zimmerman (1980) suggested that an adequate level of Hb is 10 g/dL, while 8 g/dL or b7 g/dL are borderline anemia and anemia levels, respectively. In the current study, the values of serum Hb and iron concentration were highest in the 10mg/kg ALA-supplemented treatment, indicated that iron status was improved by ALA (10 mg/kg). However, it should be noted that even though serum Hb and iron concentrations differed significantly among treatments, the values were still within an adequate range (9.62– 11.72 g/dL) during the total feeding period. Hb is formed by heme and globin, and previous research has demonstrated that heme synthesis is affected by ALA production (Cable et al., 2000); therefore, it can be deduced that ALA is related to serum hemoglobin and iron status, which is incorporate in these reactions. Serum albumin concentration differed among the treatments on day 0; this effect was not anticipated and may be due to the individual diversity of pigs in the different treatments. 4.3. Challenge responses The inflammatory and acute phase responses after challenge with endotoxin materials, such as LPS, have been well characterized in pig models (Johnson, 1997; Warren et al., 1997; Wright et al., 2000). To our knowledge, this is the first evaluation of the effect of ALA on the immune response in weanling pigs following an immunological challenge with LPS. It is commonly believed that the inflammation response is mediated by increased production of proin-
flammatory cytokines (Webel et al., 1997; Wright et al., 2000; Bosi et al., 2004). During the challenge phase, plasma cortisol, TNF-α concentrations and rectal temperature were significantly elevated in LPS treatments. These factors indicate that the challenge stimulated the pig's defense system and, therefore, presented a typical inflammation response. As reported in previous studies, antibiotics may interact with ALA, so the current study used formulated diets without antibiotics. Despite this, the pigs maintained a relatively optimal growth performance during the total feeding period. This might be largely attributed to the sanitary experimental facilities. Several studies have already indicated that a relatively clean environment, which has less potential pathogens, can decrease cytokine production compared with less sanitary facilities (Roura et al., 1991; Cromwell, 2000). From our data, it was also found that the measured parameters (rectal temperature, cortisol, IGF-I, TNF-α and haptoglobin) were similar in control pigs administered saline. On the other hand, some parameters were dramatically influenced by LPS administration. For example, cortisol and TNF-α concentrations, and rectal temperature, were lower in pigs fed the ALA diets with LPS challenge. These results agree with expectations, which indicated that inflammatory responses were alleviated in ALAsupplemented treatments and such a positive effect was probably present under pathogen challenge rather than the sanitary environment. In contrast to a previous report, IGF-I concentration was not affected by LPS injection. Hevener et al. (1997) suggested that IGF-I concentration was suppressed for 96 h after LPS injection (5 μg/kg) to finishing pigs. Wright et al. (2000) also reported an abrupt decrease in growing pigs administered LPS (100 μg/kg). Even though no LPS effect was evident in our data, an improved effect of ALA was observed at 2 h postchallenge, which response may be attribute to the growth-immune axis in response to immunological stress (Fan et al., 1995; Wright et al., 2000). Challenge stress is attenuated in pigs fed the ALA diets, with a subsequent higher IGF-I secretion. Haptoglobin is believed to be one of the acute phase proteins, related to endotoxin challenge. However, the concentration was not affected by LPS administration in the current study. This result was consistent with that of Wright et al. (2000), who reported that no elevation in haptoglobin was observed after LPS injection. Baumann and Gauldie (1994) suggested that a single dosage of LPS might not be sufficient to significant stimulate haptoglobin production. Dritz et al. (1996) also reported that repeated injection of LPS can increase haptoglobin
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concentration. Therefore, the discrepancy in the IGF-I and haptoglobin concentrations between the current and other challenge studies may be mainly due to the LPS dosage, age of pigs, route of pathogen administration or strains of E. coli used in the various experiments. It was evident from this study that exogenous ALA supplementation influenced hemoglobin and iron status in weanling pigs. Numerous studies have reported the effect of iron in maintaining resistance to infectious diseases (De Sousa et al., 1991; Brock, 1994). Hershko (1996) suggested that iron is involved in the regulation of cytokine production via its influence on second-messenger systems. Indeed, Hallquist et al. (1992) reported that bactericidal activity of macrophages was attenuated during iron deficiency. Our results also showed that immunity-related blood cell (WBC or RBC) production was positively influenced in the ALA-supplemented treatments during both feeding and challenge periods. Similar results were also reported by Mateo et al. (2006), who suggested that the RBC counts can be increased by dietary ALA supplementation. Thus, the alleviated inflammatory response found in the current study could be ascribed to elevated iron availability (heme–iron) or in circulation, and blood cell production, with a subsequent, positive influence on the immune system of piglets. 5. Conclusion In conclusion, exogenous supplementation of δaminolevulinic acid in weanling pig diets resulted in elevation of iron status and specific immune system parameters. Results suggest that δ-aminolevulinic acid has potential as a immunity enhancer in the diet of weanling pigs. This study was conducted under normal conditions (without iron deficiency); therefore, further research is necessary to evaluate the effect of δaminolevulinic acid on iron-deficient nursery pigs and its efficacy under commercial farm conditions where a greater disease challenge is present. In addition, the exact mechanism of δ-aminolevulinic acid action needs further investigation. References AOAC, 1995. Official Method of Analysis, 16th Edition. Association of Official Analytical Chemists, Washington, DC. Baumann, H., Gauldie, J., 1994. The acute phase response. Immunol. Today 15, 74–80. Beard, J.L., 2001. Iron biology in immune function, muscle metabolism and neuronal functioning. J. Nutr. 131, 568s–580s. Bosi, P., Casini, L., Finamore, A., Cremokolini, C., Merialdi, G., Trevisi, P., Nobili, F., Mengheri, E., 2004. Spray-dried plasma improves growth performance and reduces inflammatory status of weaned pigs challenged
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