Effects of purified polymannuronate on the performance, immune status, antioxidant capacity, intestinal microbial populations and volatile fatty acid concentrations of weaned piglets

Animal Feed Science and Technology 216 (2016) 161–168

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Effects of purified polymannuronate on the performance, immune status, antioxidant capacity, intestinal microbial populations and volatile fatty acid concentrations of weaned piglets W.H. Zhu a , D.F. Li b , H. Wu b , J.T. Li b , Y.Q. Chen b , H.S. Guan a,∗ , L.Y. Zhang b,∗∗ a Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China b State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China

a r t i c l e

i n f o

Article history: Received 5 January 2015 Received in revised form 21 May 2015 Accepted 22 May 2015 Keywords: Intestinal microbial Performance Piglets Polymannuronate Volatile fatty acids

a b s t r a c t The aim of this study was to assess the effects of purified, low-molecular-weight, polymannuronate on the performance, immune status, antioxidant capacity and the intestinal tract fermentation profile of weaned piglets. In a 28-d experiment, 180 crossbred Duroc × (Landrace × Yorkshire) piglets weighing 9.19 ± 1.47 kg and weaned at 35 days of age, were divided into 5 groups and fed corn-soybean meal based diets supplemented with 0, 3, 4, 5 or 6 g/kg polymannuronate (supplemented at the expense of corn). Each treatment was replicated 6 times with 6 pigs per replicate. Average daily gain (ADG), gain to feed (G:F) ratio as well as the serum IgM and hepatic glutathione linearly increased (P < 0.01) with increasing level of polymannuronate. Supplementation with increasing levels of dietary polymannuronate resulted in increased numbers of ileal and colonic lactic acid bacteria (linear effect, P < 0.01). The number of E. coli in ileum and colon was linearly decreased (P < 0.05) with increasing level of polymannuronate. At the end of the experiment, ileal and cecal lactic acid concentrations were significantly linearly increased (P < 0.01) with increasing level of polymannuronate. The concentration of butyric acid in the cecum, acetic acid and total VFA in colonic were also linearly increased (P < 0.05) with increasing polymannuronate inclusion levels. These results indicate that polymannuronate can improve immune status, antioxidant capacity and performance of weaned piglets. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Alginate is a natural acidic linear polysaccharide, extracted from brown seaweed, and is composed of (1–4) linked ␤-dmannuronic acid and (1–4) linked ␣-l-guluronic acid. Alginates exist as one of three polymers namely polymannuronate, polyguluronate, or heteropolymer (a mixture of ␤-d-mannuronic acid and ␣-l-guluronic acid residues) (Haug et al., 1967).

Abbreviations: ADG, average daily gain; ADFI, average daily feed intake; G:F, gain to feed; Mw , average molecular weight; FM , mole fraction of ␤-dmannuronate; VFA, volatile fatty acids; GLM, General Linear Model; CFU, colony-forming units. ∗ Corresponding author. Tel.: +86 532 8203 1887; fax: +86 532 8203 1887. ∗∗ Corresponding author. Tel.: +86 10 6273 1272; fax: +86 10 6273 3700. E-mail addresses: [email protected] (H.S. Guan), [email protected] (L.Y. Zhang). http://dx.doi.org/10.1016/j.anifeedsci.2015.05.015 0377-8401/© 2016 Elsevier B.V. All rights reserved.

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Alginates with a high ␤-d-mannuronic acid content have the ability to stimulate the production of cytokines such as interleukin-1, interleukin-6 and tumor necrosis factor-␣ (Otterlei et al., 1991). Son et al. (2001) found that high mannuronic acid-containing alginate can stimulate various functions of murine peritoneal macrophages, such as anti-tumor activities, phagocytosis and the production of tumor necrosis factor-␣, nitric oxide and hydrogen peroxide. Differences in the molecular weight, ratio of mannuronic acid and guluronic acid may be responsible for their diversity of bioactivities between different types of alginate. The higher content of M is, the stronger immunity and anti-tumor activity will be (Fujihara and Nagumo, 1992; Suzuki et al., 2011). Otherwise, alginate as a dietary fiber is not absorbed in the small intestine (Jonathan et al., 2013), but it may be selectively fermented by beneficial endogenous bacteria in the large intestine. Consequently, the production of short chain fatty acids may be increased in the gut which can then reduce the proliferation of exogenous bacteria (O’Sullivan et al., 2010; Patterson and Burkholder, 2003). Therefore, alginate has been proposed as a potentially useful prebiotic (O’Sullivan et al., 2010; Wang et al., 2006). Recent studies have shown the benefits of supplementing seaweed and seaweed extracts in animal diets on growth and feed efficiency. Yan et al. (2011) confirmed the favorable effects of sodium alginate oligosaccharides on growth performance, cecal microbiota composition, and mucosal immune responses in broiler chickens. It was also demonstrated that supplementation with 1–4 g/kg of dietary purified, low-molecular-weight polymannuronate can improve immune status, antioxidant capacity and performance of broiler chickens (Zhu et al., 2015). Brown seaweed extracts such as alginates, laminarins and fucoidan contain a high concentration of dietary fiber which is considered as an important factor influencing feed digestion and nutrient absorption in pigs (Walsh et al., 2013). Weight gain was greater for pigs fed diets with 1.25% high ␤-d-mannuronic acid alginate compared with the control (Gaserod and Dessen, 2011). However, purified, low-molecularweight polymannuronate has not been reported as feed additive in swine diets. Therefore, the aim of the current study was to explore the potential of polymannuronate as a novel feed additive. As weaned piglets are susceptible to gut disorders, infections and diarrhea due to social, environmental, psychological and dietary stresses (Pluske et al., 1997), they were used as the target animal to test the effects of polymannuronate on their performance, immune status, antioxidant capacity, intestinal microbial fermentation profile.

2. Materials and methods The Animal Welfare Committee of China Agricultural University (Beijing, China) approved the animal care protocol used in this experiment with the protocol reference number 20140302(2). The animal experiment was carried out in the Fengning Animal Experimental Base of the National Feed Engineering Technology Research Center (Hebei, China).

2.1. Preparation of polymannuronate The polymannuronate used in this study was provided by the National Engineering Research Center of Marine Drugs, Ocean University of China (Qingdao, China) and was produced by acid hydrolysis as described by Zhu et al. (2015). The average molecular weight (Mw ) of polymannuronate was ∼10 kDa determined by High Performance Gel Permeation Chromatography conducted on an Agilent 1100 Chromatographic Instrument (Yang et al., 2004). The purity of polymannuronate described as mole fraction of ␤-d-mannuronate (FM ) was determined to be 80% by 1 H NMR spectroscopy (Davis et al., 2003). In addition to 80% polymannuronate, the compound contains 20% of guluronic acid residues. Detailed information about the product is available from Yang et al. (2004).

2.2. Experimental diets The experiment comprised 5 dietary treatments. Diets were based on corn and soybean meal supplemented with 0, 3, 4, 5 or 6 g/kg polymannuronate (supplemented at the expense of corn). The level of polymannuronate was chosen according to the unpublished results of a pilot study on weaned piglets conducted in our laboratory. The diets were formulated to meet or exceed the requirements of 10–20 kg pigs recommended by the NRC (2012). The ingredient composition and chemical analysis of the basal diet was presented in Table 1.

2.3. Animals and management The experiment was conducted as a randomized complete block design. One hundred and eighty, crossbred Duroc × (Landrace × Yorkshire) piglets weaned at 35 days of age and weighing 9.19 ± 1.47 kg were blocked by weight, sex and litter and allotted to 1 of 5 dietary treatment. Each treatment was replicated 6 times with 6 pigs (3 males and 3 females) per replicate. All pigs were housed indoors in 1.2 × 2.1 m pens with half of the pen area being solid concrete and the remainder plastic slats. Feed and water were available ad libitum throughout the entire 28-day experiment. The room temperature was maintained at 26–28 ◦ C. Pigs and feeders were weighed individually at the beginning of the experiment, and on d 14 and d 28. These values were used to calculate ADG, ADFI and G:F ratio.

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Table 1 Composition and chemical analysis of basal diet (as-fed basis). Basal diet Ingredients (g/kg) Corn Soybean meal Extruded soybean Fish meal Whey powder Dicalcium phosphate Wheat bran Soybean oil Vitamin-mineral premixa Limestone Sodium chloride l-Lysine-HCl (78%) dl-Methionine (99%) Nutrient levelb (g/kg) ME, MJ/kg Crude protein Crude fiber Ash Calcium Total phosphorus Sodium Lysine

587 165 100 40 40 16 15 15 10 6.5 3.5 1.5 0.5 14.13 199.1 29.2 61.4 8.1 6.9 1.8 12.7

a Provided per kilogram diet: vitamin A, 9000 IU; vitamin D3 , 3000 IU; vitamin E, 20 IU; vitamin K3 , 3.0 mg; thiamine, 1.5 mg; riboflavin, 4.0 mg; pyridoxine, 3.0 mg; cobalamin, 0.2 mg; biotin, 0.1 mg; pantothenic acid, 30 mg; nicotinic acid, 15 mg; folic acid, 0.75 mg; iron (from FeSO4 ·H2 O), 75 mg; copper (from CuSO4 ), 150 mg; zinc (from ZnSO4 ), 75 mg; manganese (from MnSO4 ), 60 mg; iodine (from KI), 0.35 mg; selenium (from Na2 SeO3 ), 0.30 mg. b All values except metabolizable energy were analyzed and values are the means of two determinations.

2.4. Sampling At the end of the experiment, one pig per pen (n = 6) was randomly chosen. At 8:00 am, 9 mL blood samples were collected from the jugular vein using EDTA-K2 coated tubes and plain tubes without anticoagulant. After blood sampling on d 28, the same pigs which were bled were tattooed and shipped to the adjacent abattoir after a 12-h fasting period. Serum and plasma samples were separated by centrifugation (1342 × g) for 10 min at 4 ◦ C, and stored at −20 ◦ C until needed for analysis. After blood sampling on d 28, the same pigs which were bled were shipped for slaughter. The pigs were stunned by electric shock and then slaughtered by exsanguination. The liver and intestinal tract were collected from each of the 30 pigs at the slaughter plant at the time of evisceration. Three adjacent segments of the intestinal tract from each section (10 cm) were removed from the mid-ileum, mid-cecum and the second loop of the proximal colon by ligation at both ends for subsequent volatile fatty acids (VFA), lactic acid and microbial analyses. All samples were taken within 30 min of slaughter, and were placed in liquid nitrogen for transportation to the laboratory and then stored at −20 ◦ C until needed for analysis. 2.5. Chemical analysis Diets were analyzed according to the methods of the Association of Official Analytical Chemists (2006) for total phosphorus (AOAC Method 995.11) and calcium (AOAC Method 927.02). Crude protein was determined by the method of Thiex et al. (2002). Ash was determined after ignition in a muffle furnace (Nabertherm, Bremen, Germany) at 500 ◦ C for 4 h. Determination of crude fiber according to the intermediate filtration method (ISO 6865:2000). For lysine determination, diets were acid hydrolyzed with 6 mol/L HCl (AOAC Method 994.12) using an Amino Acid Analyzer (Hitachi L-8900, Tokyo, Japan). Antibody levels (IgG, IgM and IgA) in serum were determined by turbidimetry with commercial kits (Sanwei Biotech Firm, Weifang, China). Plasma and liver samples were analyzed for glutathione activity, glutathione peroxidase, total antioxidant capacity and the concentration of malondialdehyde. The kits used for analysis of oxidation resistance and other measures of antioxidant status were purchased from the Jiancheng Institute of Biological Engineering (Nanjing, China). The ultraviolet-visible spectrometer used was the TU-1901 produced by Puxi General Instrument Corporation (Beijing, China). The Automatic Biochemical Analyzer was a Hitachi 7160 produced by the Hitachi High-Tech Corporation (Tokyo, Japan). The concentrations of VFA in the digesta were determined by Gas Chromatography (Agilent 7890A, Agilent Technologies, Santa Clara, CA) following the procedures described by Shen et al. (2009). Lactic acid was determined by High Performance Anion Exchange Chromatography with Conductometric Detection (Dionex ICS-3000, Sunnyvale, USA) following the procedures described by Wojtczak et al. (2010).

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Table 2 Effect of graded levels of dietary polymannuronate on the performance (average daily gain (ADG), average daily feed intake (ADFI) and gain to feed (G:F) ratio) of weaned pigs (d 0–28).a Polymannuronate level (g/kg)

ADG (kg) ADFI (kg) G:F ratio (kg/kg) a

SEM

0

3

4

5

6

0.39 0.76 0.52

0.45 0.81 0.56

0.44 0.76 0.59

0.47 0.78 0.60

0.50 0.84 0.60

P-value

0.02 0.05 0.01

Linear

Quadratic

<0.01 0.11 <0.01

0.57 0.53 0.39

Values are the means of 6 replicates.

2.6. Microbiological analysis 1 mL sample of the digesta obtained from the ileum, cecum and colon was serially diluted (1:10) with 9 mL aliquots of Maximum Recovery Diluents, and spread (0.1 mL aliquots) onto selective agars. Lactic acid bacteria were isolated on de Man, Rogosa and Sharp (MRS) agar (Oxoid, Basingstoke, England) by 18–24 h incubation at 37 ◦ C in an atmosphere enriched with 5% CO2 , as recommended by the manufacturer. E. coli and total aerobic bacteria were grown on MacConkey agar and Brain Heart Infusion agar respectively, following aerobic incubation at 37 ◦ C for 18–24 h. The total anaerobic bacteria were isolated on Wilkins-Chalgren Anaerobe agar with 5% defibrinated sheep blood, and incubated 18–24 h at 37 ◦ C in an anaerobic tank (Mitsubishi Gas Chemical Company, Tokyo, Japan). All bacteria were counted and expressed as total CFU/g digesta, and the results are presented as log10 -transformed data. 2.7. Statistical analysis Statistical analyze for a randomized complete block design was performed using the General Linear Model (GLM) procedure of the SAS (SAS Inst. Inc., Cary, NC) with pen as the experimental unit. Polynomial contrasts were used to determine the linear and quadratic effects of dietary polymannuronate level. Coefficients for unequally spaced contrasts were generated by PROC IML of SAS. Results are presented as means plus SEM. Statistical significance and tendencies were set at P < 0.05 and P < 0.10 for all statistical tests. 3. Results 3.1. Performance ADG and G:F ratio increased with increasing level of polymannuronate (linear and quadratic effect, P < 0.01). There was no effect on ADFI throughout the experiment (Table 2). The inclusion of 6 g/kg polymannuronate is an optimum dose with improvements in ADG and G:F ratio throughout the duration of the experiment. 3.2. Immune status IgA and IgG were not affected (P > 0.05) by feeding diets with increasing polymannuronate inclusion levels. However, a linear (P < 0.01) effect was observed in the level of serum IgM with increasing dietary polymannuronate inclusion levels (Table 3). Table 3 Effect of graded levels of polymannuronate on immunity and antioxidant capacity of weaned pigs (d 28).a Polymannuronate level (g/kg)

Serum IgA (g/L) IgM (g/L) IgG (g/L) Total antioxidant capacity (U/mL) Glutathione (mg/L) Glutathione peroxidase (U/mL) Malondialdehyde (nmol/mL) Liver Total antioxidant capacity (U/mg) Glutathione (mg/g) Glutathione peroxidase (U/mg) Malondialdehyde (nmol/mg) a

Values are the means of 6 replicates.

SEM

0

3

4

5

6

1.11 0.73 8.21 10.2 2.20 834 7.04

1.10 0.83 8.55 9.93 2.33 820 7.06

1.15 0.84 8.68 9.53 2.21 860 6.95

1.12 0.86 8.76 10.2 2.18 870 7.31

1.17 0.95 8.60 10.2 2.21 843 7.43

1.40 0.66 53.6 0.60

1.53 0.72 49.9 0.59

1.43 0.86 50.0 0.53

1.60 1.00 53.0 0.59

1.28 0.87 50.5 0.52

P-value Linear

Quadratic

0.05 0.04 0.26 0.50 0.10 32.8 0.25

0.49 <0.01 0.85 0.97 0.84 0.56 0.28

0.70 0.44 0.32 0.38 0.50 0.97 0.34

0.11 0.06 3.33 0.03

0.86 <0.01 0.63 0.08

0.15 0.87 0.60 0.72

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Table 4 Effect of graded levels of polymannuronate on intestinal microbial populations (log10 CFU/g) of weaned pigs (d 28).a Polymannuronate level (g/kg)

Ileum E. coli Lactic acid bacteria Total aerobic bacteria Total anaerobic bacteria Cecum E. coli Lactic acid bacteria Total aerobic bacteria Total anaerobic bacteria Colon E. coli Lactic acid bacteria Total aerobic bacteria Total anaerobic bacteria a

SEM

P-value

0

3

4

5

6

3.71 5.16 5.98 5.02

3.54 5.88 6.20 6.34

2.95 6.69 6.91 7.23

3.31 6.74 6.12 6.99

2.87 6.55 5.43 6.91

0.26 0.36 0.34 0.33

0.03 <0.01 0.57 <0.01

0.80 0.46 0.02 0.08

4.24 7.83 6.86 8.38

3.80 7.71 7.64 8.02

3.51 7.61 7.57 7.89

3.68 7.90 7.92 8.06

3.42 7.52 7.48 8.03

0.30 0.22 0.35 0.22

0.06 0.52 0.10 0.23

0.76 0.89 0.27 0.36

4.42 7.31 7.90 8.37

4.60 8.52 7.93 8.33

4.28 8.45 7.65 8.26

4.09 8.23 7.95 8.22

3.50 8.26 7.50 8.15

0.25 0.14 0.20 0.13

0.02 <0.01 0.28 0.22

0.03 <0.01 0.45 0.68

Linear

Quadratic

Values are the means of 6 replicates.

3.3. Antioxidant capacity There was no effect on plasma antioxidant capacity of pigs. Hepatic glutathione increased with increasing level of polymannuronate (linear effect, P < 0.01). Hepatic malondialdehyde level showed a trend toward a linear reduction (linear effect, P = 0.08) (Table 3). 3.4. Intestinal microbiota Supplementation with increasing level of dietary polymannuronate resulted in increased ileal lactic acid bacteria, total anaerobic bacteria (linear effect, P < 0.01) and total aerobic bacteria (quadratic effect, P = 0.02). The ileal E. coli linearly declined (P = 0.03) with increasing level of polymannuronate. In the colon, the number of E. coli was significantly decreased (linear and quadratic effect, P < 0.05) and the amount of lactic acid bacteria were significantly increased (linear and quadratic effect, P < 0.01) with increasing polymannuronate inclusion levels. The number of cecal E. coli showed a trend toward a linear reduction with increased polymannuronate supplementation (linear effect, P = 0.06) (Table 4). 3.5. VFA and lactic acid Ileal lactic acid concentration increased with increasing level of polymannuronate (linear effect, P < 0.01). Ileal propionic acid tended to increase (quadratic effect, P = 0.05) in response to increasing dietary polymannuronate levels. In the cecum, the concentration of lactic acid and butyric acid raised (linear and quadratic effect, P ≤ 0.01) with increasing dietary level of polymannuronate. Dietary polymannuronate had a significant increasing effect on colonic total VFA (linear effect, P = 0.01) and acetic acid (linear effect, P < 0.01) concentrations with increasing level of polymannuronate. There were also trends toward a linear increase of colonic butyric acid (linear effect, P = 0.08) and total VAF concentration (quadratic effect, P = 0.06) with increasing polymannuronate inclusion levels (Table 5). Generally, concentrations of total VFA and major VFA (acetic and propionic acids) were in the order of colon > cecum > ileum. 4. Discussion Polymannuronate supplementation in pig diets increased ADG and improved feed conversion in the current experiment. The inclusion of 6 g/kg polymannuronate showed the greatest benefit without considering the cost of polymannuronate in growth performance with improvements in overall ADG and overall G:F ratio throughout the duration of the experiment. In agreement with this observation, Yan et al. (2011) showed that supplementation with 0.4 g/kg sodium alginate oligosaccharides resulted in higher (P < 0.01) weight in broiler chickens challenged with Salmonella enteritidis, and influenced their caecal microbiota. Likewise, pigs offered diets supplemented with laminarin had an increased ADG and gain-to-feed ratio compared to pigs offered diets without laminarin supplementation (McDonnell et al., 2010). Reilly et al. (2008) found that dietary inclusion of seaweed extracts had no effect on feed intake, weight gain, feed conversion ratio and nutrient digestibility in weanling pigs. The difference may be attributed to factors such as purity, Mw of seaweed extracts and differences in animal species. Natural seaweed fibers are predominantly high-molecular-weight polymers, so they pass through the gut too rapidly to allow bacterial utilization (Walsh et al., 2013). The polymannuronate (Mw = ∼10 kDa) used in this study is mainly composed of low-molecular-weight polymer, so its utilization efficiency is supposed to be higher than that of natural seaweed fibers used in the study of Reilly et al. (2008).

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Table 5 Effect of graded levels of polymannuronate on the concentrations (␮mol/g) of volatile fatty acids (VFA) and lactic acid in the digesta of weaned pigs (d 28).a,b Polymannuronate level (g/kg) 0 Ileum Lactic acid VFA Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Pentoic acid Total VFA Cecum Lactic acid VFA Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Pentoic acid Total VFA Colon Lactic acid VFA Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Pentoic acid Total VFA a b

3

SEM 4

5

6

P-value Linear

Quadratic

28.2

33.6

36.1

45.6

43.6

1.91

<0.01

0.28

20.0 5.27 2.14 4.28 0.90 0.91 33.5

19.2 4.02 1.76 3.61 0.62 0.81 30.0

21.4 4.80 2.24 3.75 0.93 1.08 34.2

19.9 5.37 2.18 3.90 1.01 0.97 33.3

20.8 6.52 2.04 4.21 0.71 0.72 34.9

1.32 0.79 0.20 0.40 0.20 0.25 2.07

0.65 0.32 0.94 0.78 0.91 0.84 0.56

0.84 0.05 0.56 0.17 0.86 0.61 0.24

16.8

39.9

40.0

47.0

45.9

1.75

<0.01

<0.01

50.1 16.1 2.31 9.93 0.91 0.95 80.3

51.5 18.5 2.38 13.2 0.86 1.10 87.5

51.0 13.7 2.27 12.0 0.96 1.26 81.3

52.8 13.0 2.03 13.4 0.72 0.78 82.8

55.6 14.3 2.27 13.2 0.89 0.92 87.3

2.28 1.49 0.16 0.33 0.14 0.20 3.17

0.12 0.12 0.48 <0.01 0.66 0.78 0.27

0.40 0.39 0.81 0.01 0.99 0.26 0.89

38.4

37.0

36.2

38.8

37.3

1.84

0.81

0.58

59.8 20.7 2.40 15.7 0.97 1.87 101.4

64.3 20.1 2.74 15.4 1.23 1.89 105.7

67.6 20.1 2.51 18.7 1.29 1.80 112.0

60.6 20.3 2.24 14.7 0.97 1.63 100.5

1.34 0.40 0.23 1.08 0.15 0.20 1.97

<0.01 0.15 0.29 0.08 0.90 0.75 0.01

0.13 0.81 0.64 0.21 0.68 0.21 0.06

58.8 20.9 2.69 13.6 1.10 1.60 98.7

Values are the means of 6 replicates. The concentrations of cecal VFA and lactic acid are expressed based on their original cecal content.

Many studies have shown that seaweed supplementation can enhance the immune response in livestock and improve the health of the host (O’Sullivan et al., 2010; Leonard et al., 2012). The biological activities of alginate are strongly influenced by chemical structure and molecular size. High mannuronic acid-containing alginate has immunostimulating properties on macrophages after in vivo exposure (Son et al., 2001). High-M alginate (FM = 69–86%, Mw = 30–690 kDa) showed immunological activity, while low-M alginate (M% < 31%) showed none. Alginate with Mw < 200 kDa showed the highest activity among high-M alginate (FM = 78%) (Suzuki et al., 2011). Recent studies have demonstrated that alginate oligomers as well as polymers had antioxidant activities (Tomida et al., 2010). Ueno et al. (2012) indicated that polymannuronate is capable of scavenging superoxide and hydroxyl radicals in a concentration-dependent manner. The scavenging capacity of polymannuronate with concentration 1000 ␮g/mL is higher than that of 10 and 100 ␮g/mL. Polymannuronate is also capable of stimulating RAW264.7 which is a murine macrophage-like cell line to induce nitric oxide production (Ueno et al., 2012). In our study, the immunity status and antioxidant activities of the pigs fed with polymannuronate-containing diets were both monitored and the results indicated that polymannuronate-containing diets can significantly increase the levels of serum IgM and hepatic glutathione. If smaller weaned piglets were chosen in this study, the results may be more obvious. Thus, polymannuronate of ∼10 kDa Mw (FM > 80%) can improve humoral immunity and antioxidant capacity of pigs. Because the effect of various Mw and FM on immunity and antioxidant capacity of pigs is not obtained in the present study, further study will be required. Lactic acid bacteria have potential probiotic properties and can rapidly establish a complex bacterial community to prevent colonization of pathogenic bacteria (Nazef et al., 2008). Furthermore, dietary supplementation with prebiotic compounds is a promising means to reduce pathogen shedding owing to their pathogen-inhibitory effect (Akiyama et al., 1992). Zhu et al. (2015) reported that dietary supplementation with polymannuronate can significantly decreased cecal E. coli and increased lactic acid bacteria in broilers. In the current study, the numbers of ileal and colonic lactic acid bacteria significantly increased and the colonic E. coli significantly decreased with increasing level of polymannuronate. These effects are consistent with previous reports that alginate oligosaccharides can increase the numbers of lactobacilli and bifidobacteria in vivo and in vitro while significantly decreasing the abundance of enterobacteriaceae and enterococci in feces and cecal contents of rats (Patterson and Burkholder, 2003; Akiyama et al., 1992). However, the numbers of total anaerobic bacteria and lactic acid bacteria were relatively lower than the study of Mikkelsen and Jensen, 2004 and this may be due to the different sample preparation method, medium and culture condition. Janczyk et al. (2010) reported that dietary supplementation

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with alginate resulted in a higher enterococci population in the distal small intestine, cecum and proximal colon of weanling pigs. Further molecular biological analysis of the intestinal microbiota by Denaturing Gradient Gel Electrophoresis demonstrated that pigs fed a alginate-supplemented diet had a higher microbial diversity in the distal small intestine (Janczyk et al., 2010). These results suggest that alginate supplementation can increase the number of beneficial bacteria and decrease the number of harmful bacteria. Polymannuronate in the current study should be one of active ingredients of alginate for these bioactives. Lactic acid bacteria and other beneficial bacteria can ferment carbohydrates to produce high concentrations of lactic acid and VFA, which in turn can decrease the pH of their environment and inhibit the growth of other bacteria (Olukosi and Dono, 2014; Heneghan, 1988). VFA are an important fuel for large intestinal colonocytes and can provide nutrition to intestinal epithelium, with butyric acid being the most important VFA (Heneghan, 1988). Furthermore, butyric acid can play an anti-inflammatory effect by inhibiting the formation of inflammatory cytokines (Weber and Kerr, 2008). The production of lactic acid and VFA will facilitate the growth of beneficial bacteria, improve the nutrition status and the anti-inflammatory ability of gut. The study indicates that dietary polymannuronate increased the ileal and cecal lactic acid concentrations and quadratically increased colonic total VFA and acetic acid concentrations. These results are consistent with those obtained by Reilly et al. (2008) and Lynch et al. (2010), who demonstrated that the total VFA in the colon and cecum were increased in pigs fed diets containing seaweed extracts. 5. 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