δ-Aminolevulinic acid, and lactulose supplements in weaned piglets diet: Effects on performance, fecal microbiota, and in-vitro noxious gas emissions

Livestock Science 183 (2016) 84–91

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δ-Aminolevulinic acid, and lactulose supplements in weaned piglets diet: Effects on performance, fecal microbiota, and in-vitro noxious gas emissions M.M. Hossain, J.W. Park, I.H. Kim n Department of Animal Resource & Science, Dankook University, No. 29 Anseodong, Choongnam, Cheonan 330-714, South Korea

art ic l e i nf o Article history: Received 6 February 2015 Received in revised form 22 November 2015 Accepted 26 November 2015 Keywords: Ammonia gas emissions δ -Aminolevulinic acid E. coli Hemoglobin Lactulose Weaning pigs

a b s t r a c t

δ -Aminolevulinic acid (ALA) is a non-protein amino acid that plays a rate limiting role in the process of heme biosynthesis. Lactulose (LAC) is a kind of non-digestible oligosaccharides which has been shown to improve growth performance in weaning pigs through prebiotic actions. This study evaluated the efficacy of ALA, and LAC in weaned piglets. The study was conducted with one hundred seventy five [(Yorkshire
Landrace)
Duroc] weaned piglets in a 33 d feeding trial, and one of five diets: 1) CON (basal diet, no antibiotic); 2) ALA05 (CONþ 0.5 g ALA/kg of diet); 3) ALA10 (CONþ1 g ALA/kg of diet); 4) LAC05 (CONþ0.5 g LAC/kg of diet); 5) LAC10 (CONþ1 g LAC/kg of diet). All data were statistically analyzed using the PROC MIXED procedure of SAS. Orthogonal contrasts were used to the effects of treatments. Weaning pigs fed diets with the ALA, and LAC had higher body weight (BW) compared with pigs fed the CON diet on d 19 (P ¼0.028, and 0.011), and d 33 (P ¼0.031, and 0.015), respectively. In addition, LAC supplementation had higher BW than ALA supplementation (P¼ 0.046) on d 19. Piglets fed diets with ALA, and LAC had higher average daily growth (ADG), and feed efficiency (G:F) compared with piglets fed CON diet during phase 2 (d 6–19), and overall (d 1–33), respectively (Po0.05). Besides, LAC diets improved ADG (P¼0.037), and G:F (P¼ 0.024) compared with ALA diets during phase 2. Weaned piglets fed LAC increased dry matter (DM; d 19, and 33, respectively), nitrogen (d 33), and energy (d 19) digestibility compared with those fed CON diet (Po0.05). ALA supplementation increased DM digestibility compared with CON diet (P¼ 0.041) on d 33. Piglets fed with the ALA diet increased serum total iron-binding capacity, hemoglobin, and hematocrit, and blood red blood cell compared with those fed the CON diet (Po0.05). Piglets fed with the LAC diet increased fecal Lactobacillus, and reduced E. coli counts (Po0.05) when compared with those fed the CON on d 19, and 33, respectively. Moreover, piglets fed with the LAC diets had higher fecal Lactobacillus (d 19, and 33), and lowered E. coli (d 33) than pigs those fed the ALA diets (Po0.05). The fecal moisture, and diarrhea score were not affected by dietary supplementation with ALA or LAC during the whole experiment. Piglets fed the LAC diets had reduced ammonia gas emissions compared with the CON diet on d 33 (Po0.001). In conclusion, results indicated that dietary supplementation of ALA, and/or LAC improved performance, and reduced noxious gas emissions in weaned piglets. & 2015 Elsevier B.V. All rights reserved.

1. Introduction Weaning is a critical stage for pigs because of alterations in the gastrointestinal tract structure, and function, changes to the histology, and biochemistry of the small intestine in adapting to enteric microbiota, and immune response challenges, as well as weaning stresses, such as nutritional, environmental, and social that are responsible for depressed growth performance, and nutrient malabsorption (Pluske et al., 1997; Wang et al., 2009; Zhang n

Corresponding author. E-mail address: [email protected] (I.H. Kim).

http://dx.doi.org/10.1016/j.livsci.2015.11.021 1871-1413/& 2015 Elsevier B.V. All rights reserved.

et al., 2013). Iron (Fe) is the main deficient mineral in nursery pigs due to poor efficiency of Fe transfer through the placenta (Wang et al., 2009), and low Fe concentrations in the milk of sows (NRC, 2012); furthermore, weaning pigs rapidly increase red blood cell volume, and body mass (Yu et al., 2000). Therefore, direct intramuscular (IM) injection of Fe preparations in nursery pigs has become popular in anemia prevention (Bruininx et al., 2000). Dietary δ -aminolevulinic acid (ALA) supplementation is a new approach that is expected to have beneficial effects on Fe utilization, and hemoglobin (Hb) synthesis in pigs. δ -Aminolevulinic acid plays an important role in oxygen transport through the biosynthesis of heme, which initially occurs in the mitochondrion, and involves the condensation of glycine, and succinyl CoA to form

M.M. Hossain et al. / Livestock Science 183 (2016) 84–91

ALA, and primary functional form of Fe that acts as a prosthetic group of Hb (Mateo et al., 2006; Wang et al., 2009; 2011). Based on previous study, the addition of 500 mg/kg of ALA to the diets of weaned pigs improves growth performance, serum Fe, Hb, and lymphocyte concentrations (Min et al., 2004). Our previous studies with relatively low doses (3, 10, and 50 mg/kg) suggested that ALA supplementation could affect the synthesis of Hb, and positively influence the Fe status without any significant overall growth performance in weaned pigs (Chen et al., 2008a; Wang et al., 2011; Yan and Kim, 2011 ). Lactulose (LAC) is a kind of non-digestible oligosaccharide (NDO, Schumann, 2002; Cho and Kim, 2014), which is produced by isomerization of lactose by regrouping the glucose residue to a fructose molecule. Previous studies showed that lactulose elicits a prebiotic effect by increasing Lactobacillus, and Bifidobacteria counts in pigs (Konstantinov et al., 2006). Animal studies further showed that LAC has various physiological functions including reducing fermentative diarrhea by reducing E. coli during the pig rearing period (Krueger et al., 2002), improving apparent metabolizable nitrogen, as well as reducing ammonia, and acetic acid gas emissions in broilers (Cho and Kim, 2014), and enhancing growth performance in nursery pigs, and broilers (Miguel et al., 2004; Cho and Kim, 2014; Hossain et al., 2014). However, several supplemental NDO ingredients, such as galacto-oligasaccharide (GOS), manno-oligosaccharide (MOS), and chito-oligosaccharide (COS) were shown to improve growth performance in nursery pigs via prebiotic mechanisms (Miguel et al., 2004; Yan and Kim, 2011; Zhao et al., 2012; Zhao et al., 2013). Moreover, it was previously suggested that NDO could accelerate the absorption of Fe by chelating Fe at low pH thus increasing its solubility in the intestines (Liao et al., 2007; Xia et al., 2011). Therefore, this study was designed to evaluate the effects of ALA, and LAC on performance, and fecal characteristics in weaning pigs, and examine the orthogonal contrast effect of dietary ALA versus LAC.

2. Materials, and methods All animals received human care as outlined in the guide for the care, and use of experimental animals (Dankook University, South Korea, Animal Care Committee).

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Table 1 Basal diet composition (as-fed basis). Items Ingredients (g/kg) Extruded corn Extruded oat Biscuit meal Soybean meal, 440 g crude protein/kg Fermented soybean Fish meal Soy oil Lactose Whey Milk product MCP Sugar Plasma powder L-Lys HCl DL-Met L-Thr Choline chloride Vitamin premixb Trace mineral premixc Limestone Salt Calculated values (g/kg) ME (MJ/kg) SIDd Lysine SID Methionine SID Tryptophan SID Threonine Ca Avail P Analyzed values (g/kg) pH CP Ca Total P Crude fat

Phase 1a

Phase 2a

Phase 3a

111.5 100.0 – 80.0 78.0 50.0 41.5 100.0 165.0 130.0 12.5 40.0 65.0 1.2 2.6 7.7 2.0 1.0 2.0 – –

349.2 – 50.0 200.0 82.0 40.0 48.0 60.0 100.0 20.0 10.0 20.0 – 2.5 1.5 0.8 1.0 1.0 2.0 2.0 2.0

451.0 – 90.0 296.5 – 25.0 30.0 –

14.8 14.8 4.7 2.7 7.8 8.1 5.5

14.8 14.7 4.7 2.7 7.7 7.8 5.5

14.6 13.4 4.2 2.8 6.8 7.5 4.3

6.05 219.3 8.1 7.5 51.1

6.02 210.1 7.7 7.6 50.1

6.03 204.7 7.4 7.4 39.8

20.0 6.0 – – 1.6 1.4 – 1.0 1.0 2.0 3.0 3.0

a Phase 1, provided during 1–5 days; phase 2, provided during 6–19 days, and phase 3, provided during 20–33 days. b Provided per kilogram of diet: 15,000 IU of vitamin A, 3,750 IU of vitamin D3, 37.5 mg of vitamin E, 2.55 mg of vitamin K3, 3 mg of thiamin, 7.5 mg of riboflavin, 4.5 mg of vitamin B6, 24 μg of vitamin B12, 51 mg of niacin, 1.5 mg of folic acid, 0.2 mg of biotin, and 13.5 mg of pantothenic acid. c Provided per kilogram of diet: 37.5 mg of Zn, 37.5 mg of Mn, 37.5 mg of Fe, 3.75 mg of Cu, 0.83 mg of I, 62.5 mg of S, and 0.23 mg of Se. d SID ¼ Standardized ileal digestible.

2.1. Animals, and diets A total of 175 crossed healthy weaning pigs [(Yorkshire
Landrace)
Duroc] with an average body weight (BW) of 8.047 0.92 kg (28d of age) were used in a 33 days experiment. Pigs were randomly allotted to 1 of 5 experimental diets according to initial BW in a randomly complete block design. There were 7 replicated pens per treatment with 5 pigs (3 gilts, and 2 borrows) per pen. Dietary treatments were: 1) CON: basal diet, no antibiotic 2) CONþ0.5 g ALA/kg of diet (ALA05), 3) CONþ1 g ALA/ kg of diet (ALA10), 4) CONþ 0.5 g LAC/kg of diet (LAC05), and 5) CONþ1 g LAC/kg of diet (LAC10). The experiment included 3 phases (5-14-14 day phase feeding; phase 1, provided during d 1 to 5; phase 2, provided during d 6 to 19, and phase 3, provided during d 20 to 33). All diets were formulated to meet or exceed the recommendation of NRC (2012) for weaning pigs, and were fed in a mash form (Table 1). ALA, and LAC as powder form were supplied by EasyBio, Seoul, Korea, and added at 0.5, and 0.1 g/kg to the basal diet, respectively. All pigs were housed in an environmentally controlled nursery room. The stainless steel pens were 0.5 m
0.6 m
2.0 m with a slatted plastic floor. Each pen was provided with a stainless steel feeder and a nipple waterer that allowed ad libitum access to feed and water throughout the experiment. Ventilation was provided by a mechanical system and

lighting was automatically regulated to provide 12 h of artificial light per day. The ambient temperature within the room was approximately 30 °C at the start of the experiment and decreased by 1 °C each wk.

2.2. Chemical analysis Feed, and fecal samples were ground to pass through a 1-mm screen, after which they were analyzed for DM (method 934.01; AOAC, 2000), crude protein (CP, method 990.03; AOAC, 2000), crude fat (method 920.39; AOAC, 1995), calcium (Ca, method 984.01; AOAC, 1995), and phosphorus (P, method 965.17; AOAC, 1995). Nitrogen (N) was determined (Kjeltec 2300 Nitrogen Analyzer; Foss Tecator AB, Hoeganaes Sweden), and CP was calculated as N
6.25. Gross energy (GE) was analyzed by oxygen bomb calorimeter (Parr 1600 Instrument Co., Moline, IL, USA). The pH of the feed sample was measured by a calibrated, glass electrode pH meter (WTW pH 340-A, WTH Measurement Systems, Ft. Myers, FL, USA).

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2.3. Sampling, and measurements Individual pig BW, and feed refusals were recorded on d 5, 19, and 33 to calculate average daily gain (ADG), average daily feed intake (ADFI), and growth efficiency (G:F). All pigs were fed diets mixed with 2 g/kg of chromium oxide (Cr2O3) as an indigestible marker during d 13–19, and d 27–33. Fresh fecal grab samples were randomly collected from 1 gilt, and 1 barrow in each pen (d 17–19, and d 31–33, respectively) via rectal massage. Representative samples were stored at 20°C until analyzed. Before chemical analysis, the fecal samples were thawed, and dried at 60 °C for 72 h, after which they were finely ground to a size that could pass through a 1-mm screen. All feed, and fecal samples were then analyzed for DM, N, and energy (E) as described before. Chromium was analyzed using UV absorption spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan). The apparent total tract digestibility (ATTD) was then calculated according to the method described by Zhao et al. (2013), and using the following formula: digestibility (%) ¼{1  [(Nf
Cd)/(Nd
Cf)]}
100, where, Nf¼ nutrient concentration in feces (% DM), Nd ¼nutrient concentration in diet (% DM), Cd ¼chromium concentration in diet (% DM), and Cf ¼chromium concentration in feces (% DM). The incidence of diarrhea in weaning pigs was observed, and recorded 3 times per day throughout the study. In order to assess the severity of diarrhea, feces from each pen were scored according to the method of Zhou et al. (2012). In brief, the scores were as follows: 1 (hard, dry pellets in a small, hard mass). 2 (hard, formed stool that remains firm, and soft) 3 (soft, formed, and moist stool that retains its shape), 4 (soft, unformed stool that assumes the shape of the container), and 5 (watery, liquid stool that can be poured). A cumulative diarrhea score (DS) per diet, and day was then assessed (Smiricky et al., 2002). For the blood profiles, 2 pigs from each pen (1 gilt, and 1 barrow) were randomly selected, and blood samples were collected via jugular venipuncture on d 19, and 33, respectively. Half of the sample was transferred into either a vacuum (clot activator with gel) or a 5 ml K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). Blood samples were centrifuged at 3000
g for 15 min to recover serum, which was stored at –20 °C until further analysis for Fe, total iron-binding capacity (TIBC), Hb, and hematocrit (HCT) using an automatic biochemistry blood analyzer (HITACHI 747; Hitachi, Tokyo, Japan). The red blood cell (RBC) counts of the whole blood samples were determined using an automatic blood analyzer (ADVIA 120; Bayer, Tarrytown, NY, USA) according to the method described by Wang et al. (2011), and Hossain et al. (2015). Fecal samples were collected directly by massaging the rectum of 2 pigs randomly selected from each pen (1 gilt, and 1 barrow) on d 19, and 33, and pooled, and transported to the laboratory, where microbial analysis were immediately carried out according to the method described by Hossain et al. (2015). The obtained fecal sample (1 g) from each pen was diluted with 9 mL of 10 g/L peptone broth (Becton, Dickinson, and Co., Rutherford, NJ, USA), and homogenized. Viable counts of bacteria in the fecal samples were then conducted by plating serial 10-fold dilutions (in 10 g/L peptone solution) onto MacConkey agar plates, Rappaport Vassiliadis broth plates (Neogen Corporation), and lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to isolate the E. coli, Salmonella, and Lactobacillus, respectively. The lactobacilli medium III agar plates, MacConkey agar plates, and Rappaport Vassiliadis broth (Neogen Corporation) plates were then incubated for 48 h at 39 ◦C, 24 h at 37 ◦C, and 48 h at 42 °C under anaerobic conditions, respectively. The E. coli, Salmonella, and Lactobacillus colonies were counted immediately after removal from the incubator. For analysis of the fecal ammonia (NH3), and hydrogen sulfide

(H2S), 200 g of fresh feces were randomly collected from 2 pigs (1 gilt, and 1 barrow) in each pen on d 19, and 33 of the experiment according to the method described by Zhao et al. (2013). The total fresh sampled feces were then stored in 2.6-L plastic boxes with a small hole in the middle of one side that was sealed with adhesive plaster. The samples were allowed to ferment for 7 days at room temperature (25 °C). After the fermentation, a gas sampling pump (Model GV-100; Gastec Corp., Avase, Japan) was utilized for gas detection (Gastec detector tube No. 3La for NH3, and No. 4LK for H2S; Gastec Corp.) Before measurement, the fecal samples were manually shaken for approximately 30 s to disrupt any crust formation, and to homogenize the samples, and then 100 mL of the headspace air was sampled from approximately 2.0 cm above the sample. 2.4. Statistical analysis All data were statistically analyzed using the repeated measure s statement of the PROC MIXED procedure of SAS/STAT 9.4 (SAS, 2013) when pig effect, and time effect were part of the model for a randomized complete block design. Data on growth performance, nutrient digestibility, and diarrhea score were based on a pen basis, whereas data on blood profiles, fecal microbial shedding, and noxious gas emission were based on individual pig. Before conducting statistical analysis of the Lactobacillus, E. coli, and Salmonella counts, the value was transformed logarithmically. Orthogonal contrasts were used to the effects of treatments: CON vs. ALA, CON vs. LAC, ALA vs. LAC, ALA05 vs. ALA10, and LAC05 vs. LAC10 treatments. Variability in the data was expressed as the pooled SE, and differences were deemed significant when Pr 0.05, and trends were noted when 0.05 oP o0.1.

3. Results 3.1. Growth performance Weaning pigs fed diets with the ALA, and LAC had higher BW than pigs fed the CON diet on d 19 (P¼ 0.028, and 0.011), and d 33 (P ¼0.031, and 0.015), respectively (Table 2). Moreover, LAC supplementation had higher BW, as compared with the ALA (P ¼0.046) on d 19. Within the ALA supplementation group, ALA10 diet had improved BW, as well as a trend towards an increase in BW (P ¼0.027, and 0.056), as compared with the ALA05 on d 19, and 33, respectively. No differences were observed in ADG, ADFI, and G:F among dietary treatments during phase 1 (d 1–5; P4 0.10). Weaning pigs fed on diets with ALA, and LAC had higher ADG, and G:F (P o0.05) than pigs fed the CON diet during phase 2 (d 6–19), and overall (d 1–33), respectively. Moreover, LAC supplementation improved ADG (P ¼0.037), and G:F (P ¼0.024), as compared with the ALA diet during phase 2. The ALA10 diet increased G:F (P ¼0.028), as compared with pigs fed the ALA05 diet for the same period. Weaning pigs fed the LAC diet had higher ADG (P ¼0.026), and along with the ALA diet tended to increase ADG, as compared with those fed the CON diet (P¼ 0.084) during phase 3. 3.2. ATTD of nutrients Pigs fed LAC increased DM, and E digestibility, as compared with those fed the CON diet on d 19 (P¼ 0.010, and 0.015, respectively; Table 3). During this time, pigs fed the ALA, and LAC diets tended to increase DM, and N digestibility, as compared with those fed the CON diet, respectively (P¼ 0.084, and 0.092). Moreover, pigs receiving diet supplemented with LAC had higher DM, and N digestibility than pigs fed the CON diet on d 33(P ¼0.023,

M.M. Hossain et al. / Livestock Science 183 (2016) 84–91

87

Table 2 The effects of δ-aminolevulinic acid, and lactulose on growth performance in weaning pigsa. Treatmentsb

Phase 1d (g)

BW

Phase 2d(g)

Phase 3d(g)

Overalld (g)

d1

d5

d 19

d 33

ADG

ADFI

G:F

ADG

ADFI

G:F

ADG

ADFI

G:F

ADG

ADFI

CON

8.05

9.13

14.58

20.87

216

263

0.816

389

622

0.624

449

705

0.639

393

609

ALA05

8.04

9.14

14.80

21.31

219

264

0.823

405

631

0.642

465

707

0.657

407

614

ALA10

8.04

9.31

15.43

22.24

253

303

0.830

437

642

0.682

486

730

0.668

435

634

LAC05

8.05

9.18

15.27

21.94

226

273

0.825

435

639

0.680

476

721

0.661

426

624

LAC10

8.05

9.34

15.76

22.65

259

309

0.837

459

654

0.702

492

724

0.680

447

637

SEc

0.01

0.08

0.19

0.33

17

16

12

11

12

14

10

10

G:F

0.645 0.663 0.687 0.681 0.702 0.02

0.01

0.02

0.01 P-value CON VS. ALA CON VS. LAC ALA VS. LAC ALA05 VS. ALA10 LAC05 VS. LAC10

– –

0.393 0.237

0.028 0.011

0.031 0.015

0.352 0.223

0.311 0.180

0.668 0.548

0.034 0.015

0.268 0.071

0.017 0.010

0.084 0.026

0.440 0.321

0.226 0.100

0.020 0.014

0.208 0.072

0.028 0.013

– –

0.678 0.175

0.046 0.027

0.127 0.056

0.716 0.177

0.674 0.105

0.833 0.809

0.037 0.059

0.365 0.459

0.024 0.028

0.486 0.217

0.782 0.272

0.613 0.637

0.129 0.055

0.479 0.148

0.124 0.125

–

0.180

0.076

0.136

0.181

0.128

0.681

0.170

0.336

0.213

0.367

0.886

0.414

0.135

0.346

0.177

a Dietary treatments were as follows: CON: basal diet, no antibiotic; CONþ 0.5 g ALA/kg of diet (ALA05); CONþ1 g ALA/kg of diet (ALA10); CONþ 0.5 g LAC/kg of diet (LAC05), and CONþ 1 g LAC/kg of diet (LAC10). b There were seven replication pens of five pigs/pen per treatment. c Pooled SE. d Unit in each phase is gram (g) except G:F.

Table 3 The effects of δ-aminolevulinic acid, and lactulose on nutrient digestibility (%) in weaning pigsa. Treatmentsb

CON ALA05 ALA10 LAC05 LAC10 SEc P-value CON VS. ALA CON VS. LAC ALA VS. LAC ALA05 VS. ALA10 LAC05 VS. LAC10

d 19

d 33

DM

N

E

DM

N

E

80.36 82.00 83.31 83.84 84.12 1.05

79.35 81.12 81.37 81.82 81.65 1.12

81.09 82.24 83.43 84.44 84.73 1.16

79.52 81.71 82.29 82.13 82.79 1.10

77.06 77.93 78.41 80.34 81.12 1.15

80.56 81.01 81.98 81.28 82.11 1.24

0.084 0.010

0.177 0.092

0.203 0.015

0.041 0.023

0.435 0.014

0.545 0.463

0.216 0.387

0.664 0.878

0.122 0.450

0.648 0.680

0.034 0.769

0.874 0.577

0.849

0.916

0.854

0.643

0.633

0.646

a Dietary treatments were as follows: CON: basal diet, no antibiotic; CONþ 0.5 g ALA/kg of diet (ALA05); CONþ 1 g ALA/kg of diet (ALA10); CONþ0.5 g LAC/kg of diet (LAC05), and CONþ1 g LAC/kg of diet (LAC10). b There were two pigs/pen per treatment. c Pooled SE.

and 0.014, respectively). In this period, ALA supplementation increased DM digestibility, as compared with the CON diet (P ¼0.041). Besides, LAC supplementation had higher N digestibility, as compared with the ALA diet (P¼ 0.034). Energy digestibility was unaffected by the dietary treatments on d 33 (P4 0.10). 3.3. Blood profiles, and fecal characteristics Piglets fed with the ALA diet had increased (P o0.05) serum TIBC, Hb, and HCT, and blood RBC, as compared with those fed the CON diet (Table 4). No differences were observed in Fe among the treatments throughout the experiment; however, there was a trend towards increased serum Hb with the ALA supplementation,

as compared with the LAC supplementation on d 33 (P ¼0.079). Weaning pigs fed with the LAC diet increased Lactobacillus, and reduced E. coli counts (Po 0.05), as compared with those fed the CON, and ALA diets on d 19 (Table 5). However, pigs fed the ALA diet had no significant differences in E. coli, Salmonella, and Lactobacillus counts when compared with those fed the CON diet on d 19, and 33(P 40.10). On d 33, pigs fed with the LAC diet had improved Lactobacillus counts, as compared with the CON, and ALA diets, respectively (P o0.05); moreover, LAC supplementation resulted in lower fecal E. coli than pigs fed the CON diet (P o0.001). There was a trend towards reduced fecal E. coli with the LAC supplementation (P ¼0.062), as compared with the ALA supplementation. Fecal moisture, and DS were not affected by dietary supplementation with ALA or LAC during the entire experiment (Table 6). Pigs fed the LAC diet had lowered NH3 gas emission, as compared with the CON diet on d 33 (P o0.001). There was a trend towards reduced fecal NH3 gas emission with LAC10 supplementation (P ¼ 0.089), as compared with the LAC05 supplementation. Despite the lowered fecal NH3 gas emission, H2S was unaffected by dietary treatments.

4. Discussion 4.1. Effects of ALA, and LAC in weaned piglets Recently, bacterial fermentation of ALA has been developed with reasonable cost, facilitating ALA administration in animal studies. However, there are limited studies of ALA on performance in weaned pigs. In the current study, we found a beneficial effect on the growth performance (BW, ADG, and G:F) when pigs were fed ALA (0.5 g, and/or 1 g/kg) supplemental diet during phase 2, and overall, as compared with the CON diet. These results are in agreement with one of our previous studies (Chen et al., 2008a) that reported that the supplementation of 15 mg/kg ALA increased the growth performance in weaned pigs in phase 3; but no such effect for the overall period, likely due to the effect of increased

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Table 4 The effects of δ-aminolevulinic acid, and lactulose on blood profiles in weaning pigsa. Treatmentsb

CON ALA05 ALA10 LAC05 LAC10 SEc P-value CON VS. ALA CON VS. LAC ALA VS. LAC ALA05 VS. ALA10 LAC05 VS. LAC10

Fe (μg/dL)

RBC (106/mL)

TIBC (μg/dL)

Hb (g/dL)

HCT (%)

d 19

d 33

d 19

d 33

d 19

d 33

d 19

d 33

d 19

d 33

18.0 19.9 21.7 18.4 19.3 2.2

27.1 34.4 38.9 29.3 31.3 4.7

611 648 652 630 648 20

655 713 714 677 697 19

3.8 4.9 5.4 4.5 4.7 0.7

6.1 6.9 7.1 6.6 6.6 0.3

5.8 6.2 6.3 6.1 6.2 0.6

7.2 8.0 8.2 7.5 7.6 0.3

21.2 23.1 23.3 22.4 22.5 3

26.7 30.6 30.8 28.3 29.8 1.2

0.400 0.758

0.113 0.591

0.114 0.249

0.021 0.184

0.106 0.345

0.042 0.224

0.483 0.598

0.025 0.379

0.597 0.741

0.014 0.135

0.511 0.402

0.189 0.513

0.577 0.887

0.185 0.959

0.388 0.598

0.283 0.701

0.829 0.892

0.079 0.633

0.808 0.974

0.194 0.909

0.790

0.767

0.526

0.460

0.881

0.924

0.946

0.918

0.982

0.392

a

Dietary treatments were as follows: CON: basal diet, no antibiotic; CONþ 0.5 g ALA/kg of diet (ALA05); CONþ1 g ALA/kg of diet (ALA10); CONþ 0.5 g LAC/kg of diet (LAC05), and CONþ 1 g LAC/kg of diet (LAC10). b There were two pigs/pen per treatment. c Pooled SE.

Table 5 The effects of δ-aminolevulinic acid, and lactulose on fecal microbiota (log10 cfu/g of wet digesta) in weaning pigsa. Treatmentsb

d 19 Lactobacillus

CON ALA05 ALA10 LAC05 LAC10 SEc P-value CON VS. ALA CON VS. LAC ALA VS. LAC ALA05 VS. ALA10 LAC05 VS. LAC10

d 33 E. coli

Salmonella

Lactobacillus

E. coli

Salmonella

7.36 7.47 7.46 7.60 7.65 0.06

6.50 6.48 6.47 6.43 6.41 0.03

2.41 2.42 2.34 2.36 2.40 0.05

7.38 7.42 7.47 7.56 7.60 0.03

6.51 6.47 6.45 6.43 6.39 0.03

2.39 2.42 2.44 2.39 2.43 0.04

0.441 o 0.001

0.282 o 0.001

0.690 0.715

0.115 o0.001

0.149 o 0.001

0.479 0.754

0.039 0.382

0.028 0.654

0.966 0.283

0.017 0.840

0.062 0.676

0.626 0.805

0.531

0.824

0.554

0.473

0.385

0.433

a Dietary treatments were as follows: CON: basal diet, no antibiotic; CONþ 0.5 g ALA/kg of diet (ALA05); CONþ1 g ALA/kg of diet (ALA10); CONþ 0.5 g LAC/kg of diet (LAC05), and CONþ 1 g LAC/kg of diet (LAC10). b There were two pigs/pen per treatment. c Pooled SE.

DM, and N digestibility during phase 3, on growth performance. Similarly, in this study ALA supplementation improved DM digestibility during phase 3, this might explain the positive effect on growth performance. However, it could not be ascertained clearly why DM digestibility was improved by ALA supplementation in the previous, and current study. Conversely, no significant differences were observed in response to the addition of ALA up to 50 mg/kg in weaning pigs (Mateo et al., 2006; Chen et al., 2008a), and broilers (Chen et al., 2008b). The discrepancies in results might be due to species effects, dose effects, and sanitary conditions employed during the experiment (Mateo et al., 2006). Several researchers have reported that prebiotics have a positive effect on growth performance without adverse effects on mortality in pigs, and chickens (Zhou et al., 2012; Cho and Kim, 2014). In the current study, pigs fed with the LAC diet improved BW (d 19, and d 33), ADG (phase 2, phase 3, and overall), and G:F (phase 2, and overall) than those fed with the CON diet, which is in agreement with Krueger et al. (2002) and Hossain et al. (2014) who reported that LAC supplementation in diets for sows, and weaning pigs improved ADG, respectively. Several researchers also

demonstrated that other prebiotics (i.e. MOS, COS, and FOS) supplementation showed similar growth performance, as compared with the CON diet in pigs, and broilers (Estrada et al., 2001; Li et al., 2007; Zhao et al., 2012; Zhou et al., 2012; Zhao et al., 2013). It has been suggested that the improved growth performance observed in response to dietary lactulose supplementation occurs through an increased nutrient digestibility (Tang et al., 2005). Inclusion of LAC improved DM, N, and E digestibility, as compared to the CON diet, which is in agreement with our previous studies (Zhao et al., 2012; Cho and Kim, 2014; Hossain et al., 2014) that indicated an increased nutrient digestibility in broilers, and weaned piglets in response to 1–10g/kg of lactulose supplementation. Increased nutrient digestibility may be due to improved nutrient absorption (Shim et al., 2005; Zhao et al., 2013; Zhang et al., 2013). Moreover, Tang et al. (2005) reported that NDO supplementation increased villus height (VH), and villus height to crypt depth (VH:CD) that are responsible for increased nutrient absorption. In the current study, dietary ALA increased serum RBC, Hb, HCT, and TIBC in weaned pigs. Previous studies reported that 10 mg/kg,

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89

Table 6 The effects of δ-aminolevulinic acid, and lactulose on diarrhea score, fecal moisture, and noxious gas emission in weaning pigsa. Treatments

CON ALA05 ALA10 LAC05 LAC10 SEc P-value CON VS. ALA CON VS. LAC ALA VS. LAC ALA05 VS. ALA10 LAC05 VS. LAC10

DS

Fecal moisture (%)

NH3 gas emission (mg/kg)b

H2S gas emission (mg/kg)b d 19

d 33

26.9 27.1 26.4 27.6 26.0 1.8

27.6 27.1 26.1 27.2 27.3 1.9

d 19

d 33

d 19

d 33

d 19

d 33

3.1 3.09 3.09 3.10 3.09 0.02

3.11 3.13 3.12 3.11 3.10 0.03

71.06 71.57 71.17 71.14 70.90 2.04

70.88 70.65 71.04 70.68 70.67 2.42

17.8 16.3 16.8 15.8 15.3 0.7

0.641 0.815

1.000 0.820

0.902 0.988

0.989 0.946

0.345 0.751

0.751 o 0.001

0.971 0.758

0.698 0.881

0.775 1.000

0.781 0.433

0.866 0.889

0.946 0.946

0.465 0.654

0.364 0.451

1.000 0.778

0.769 0.718

0.686

0.694

0.937

0.998

0.541

0.089

0.536

0.959

18.3 17.8 17.3 16.8 14.1 1.2

a

Dietary treatments were as follows: CON: basal diet, no antibiotic; CONþ 0.5 g ALA/kg of diet (ALA05); CONþ1 g ALA/kg of diet (ALA10); CONþ 0.5 g LAC/kg of diet (LAC05), and CONþ 1 g LAC/kg of diet (LAC10). b 200 g fresh feces for each replicate pen. c Pooled SE.

and 5 mg/kg ALA improved serum Hb, and Fe concentration in weaning pigs (Chen et al., 2008a; Wang et al., 2009), and laying hens (Chen et al., 2008c), respectively. It has been reported that supplementation with exogenous ALA can bypass such feedback regulation, thereby enabling the production of additional heme (Mateo et al., 2006). Thus, the positive effect on RBC in the current study could be ascribed to the increased Fe availability (hemeiron) or blood cell production. Mateo et al. (2006) also reported that 500 mg/kg of ALA supplementation increased RBC concentration in nursery pigs. Hematocrit is the percentage of blood that consists of red blood cells. Results of previous studies indicated that the exogenous addition of ALA increases the HCT values (Yu et al., 2000). Total iron-binding capacity (TIBC) indicates the maximum amount of Fe needed to saturate (protein can carry iron in the blood) plasma or serum transferrin (TRF), which is the primary iron-transport protein (Aisen et al., 1966). Similar with the current result, Min et al. (2004), and Chen et al. (2008c) also demonstrated that dietary supplementation of 2 g/kg, and 15 mg/kg increased TIBC concentration in weaned pigs, and laying hens, respectively. However, the inclusion of ALA did not significantly affect the Fe concentration in this study. Min et al. (2004) reported that dietary supplementation of 2 g/kg significantly increased Fe concentration in weaned pigs. ALA levels (Yan and Kim, 2011), Fe deficiency (Underwood and Suttle, 1999), and living bacteria influence the release of Fe from heme or Hb liberated from dying cells (Law et al., 1992), which may explain the inconsistent results. In our current study, the hematological characteristics (i.e. RBC, Hb, Fe, HCT, and TIBC) were unaffected by dietary LAC supplementation, which is consistent with our previous study (Cho and Kim, 2014) that suggested no positive effect on blood parameters in broilers on 1 g/kg or 2 g/kg LAC supplementation. Moreover, according to Schumann (2002) most dietary lactulose is absorbed; however, a slight amount (2.5–20 g/kg) of dietary LAC could be absorbed into the cardiovascular system that might have led to insignificant hematological results in the current study. However, no study has investigated the effect of LAC on blood parameters in weaning pigs. Therefore, further study is still necessary to investigate the effect of LAC supplementation. Pigs fed LAC supplemented diets improved Lactobacillus (d 19, and d 33), and reduced E. coli (d 19, and d 33), as compared with CON diet, which is in agreement with Bailey et al. (1991), who suggested that supplementation of 4 g/kg FOS in the diet of broiler chicks significantly increased the number of Bifidobacteria, and

Lactobacillus, and decreased E. coli in the cecum, and small intestine. Moreover, Cho and Kim (2014) have suggested that lactulose inclusion in the basal diet can reduce E. coli, and improve the Lactobacillus counts in the broiler. Several studies on broilers, and rats suggested that the inclusion of lactose for the production of lactic acid in the ceca could result in predominantly beneficial bacteria, such as Lactobacillus, and Bifidobacteria by suppressing undesirable bacteria, such as Salmonella, and E. coli. (Patterson and Burkholder, 2003; Zhao et al., 2013; Cho and Kim, 2014), which could explain the increased Lactobacillus, and decreased E. coli in this study. In the current experiment, in-vitro NH3 gas emission was reduced by the addition of LAC to the diet at 0.05 or 1 g/kg, which is in agreement with Cho and Kim (2014), who found the same result in broiler. Previous studies have observed that fecal nitrogen excretion, noxious gas, and volatile fatty acid production can be reduced by NDO supplementation (Zhou et al., 2012; Zhao et al., 2012; Zhao et al., 2013). Flickinger et al. (2003) determined that FOS supplementation (1.9 g/d) tended to decrease fecal NH3 concentration in dogs. Terada et al. (1992) reported that dietary supplementation with lactosucrose decreased fecal concentration of odor components (ammonia, indole, ethylphenol, phenol, skatole, and butyric acid) in dogs. The possible reasons for the reduction in NH3 gas emissions may be attributable to the beneficial effects on the Lactobacillus populations present in the large intestine, as the inclusion of dietary non-starch polysaccharides promotes carbohydrate-fermenting bacteria (O'Connell et al., 2005). However, ALA supplementation did not affect the fecal microbiota, and gas emission in the present study. Fecal noxious gas emission is reportedly related to intestinal microbiota (Ferket et al., 2002; Zhao et al., 2013). Several studies also suggested that the fecal noxious gas content was related to the digestibility of nitrogen (Zhang et al., 2013; Zhao et al., 2013) because the increased digestibility may allow less substrate for microbial fermentation in the large intestine with consequent decrease in the fecal noxious gas content. In this study, ALA supplementation increased DM digestibility during phase 3 without any change in N, and E digestibility that can support our present findings. To the best of our knowledge, there is no comparative data on the effect of ALA supplementation in pigs; thus, further studies will be necessary to evaluate the effects of ALA on fecal microbiota, and gas emission.

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4.2. Effects of ALA versus LAC in weaned pigs Nutrient digestibility, the immune system, and gut health are positively correlated with growth performance (Zhang et al., 2013; Zhao et al., 2013). The reasons for improved growth performance by ALA, and LAC supplementation may be explained by our present findings on nutrient digestibility, hematological parameters, and fecal microbiota. Moreover, LAC supplementation improved BW (d 19), ADG (d 19), and G:F (d 19), as compared with ALA supplementation. The above findings may be due to improved Lactobacillus (d 19, and d 33), and reduced E. coli (d 19), and N digestibility observed on d 33. Previous studies had shown that diets rich in prebiotic carbohydrates – for example, fructan, lactose, lactulose, and FOS– improved the stability of the intestinal microbial flora, and subsequently improved the growth performance of newly weaned piglets, and poultry (Konstantinov et al., 2006; Zhou et al., 2012; Zhao et al., 2013; Cho and Kim, 2014; Hossain et al., 2014). ALA supplementation tended to improve Hb (d 33), as compared with LAC supplementation. 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, ALA possibly regulates serum Hb, and Fe status, since a reaction limiting enzyme, ALA synthase, is modulated by the production of heme via a feedback mechanism in most mammalian cells (Jover et al., 2000). On the other hand, a clinical study reported that ingestion of NDO effectively improves serum Hb levels in patients with chronic renal failure (Jing et al., 1997). LAC can increase Fe absorption by chelating, and increasing solubility in the gastrointestinal tract, however no positive effect was observed in the current study.

5. Conclusion In conclusion, both ALA, and LAC (0.5, and/or 1 g/kg, respectively) supplementation increased growth performance and ATTD of nutrients in weaning piglets. Moreover, diet with ALA supplementation increased blood parameters (i.e. Hb, TIBS, and HCT, and RBC), whereas LAC supplementation improved gut health and reduced fecal NH3 gas emissions. These results indicated that ALA, and LAC supplementation might have a positive role on performance through Hb biosynthesis, and prebiotic mechanisms in nursery piglets, respectively.

Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of the manuscript entitled, 'δ-aminolevulinic acid, and lactulose supplements in weaned piglets diet: Effects on performance, fecal microbiota, and in-vitro noxious gas emissions'.

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