Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets

Livestock Science ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets Xiangfang Zeng a, Zhimin Huang a, Fengrui Zhang a, Xiangbing Mao b, Shihai Zhang a, Shiyan Qiao a,n a b

State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural University, Beijing, People’s Republic of China Animal Nutrition Institute, Sichuan Agricultural University, Key Laboratory of Animal Disease-Resistance Nutrition, Ministry of Education, Ya’an, People’s Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 27 April 2015 Received in revised form 14 August 2015 Accepted 7 September 2015

The objective of this study was to investigate the effects of oral N-carbamylglutamate (NCG) administration on intestinal digestive enzymes, microbiota population and immunity under physiological condition in newborn piglets. A total of 48 1-d-old piglets (sow reared) were allotted, based on the initial body weights (1.57 70.04 kg), into 4 treatments (12 piglets/group): (1) 0.52 g/kg body weight (BW) LAlanine (control), (2) 0.31 g/kg BW L-Arginine
HCl (arginine), (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG, and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW NCG. The supplement were offered twice daily for 14 d. At d 7 and d 14, 6 piglets from each group were killed. Piglets supplemented with 50 mg/ kg BW NCG had increased average daily gain during days 1–14 after birth and jejunal mucosal lactase activity on d 7 compared with piglets supplemented with control, arginine and 100 mg/kg BW NCG groups. Serum concentrations of arginine, glutamate, citrulline and ornithine in arginine, 50 and 100 mg/ kg BW NCG groups were markedly increased at d 7 and 14, in comparison with the control group (P o0.05). Moreover, the number of cecal Lactobacillus spp. in both NCG groups was higher than the control and arginine groups at d 14 (Po 0.05). Serum IgA (P ¼0.05) and ileal sIgA (P ¼0.08) concentrations in the 50 mg/kg BW NCG and arginine groups were higher than those in the control and 100 mg/kg BW NCG groups at d 14. In conclusion, oral administration of 50 mg/kg BW NCG to newborn piglets improved growth performance, increased the endogenous synthesis of arginine and arginine family of amino acids, jejunal lactase activity and cecal Lactobacillus spp. numbers, and stimulated secretion of ileal sIgA. & 2015 Published by Elsevier B.V.

Keywords: N-carbamylglutamate Suckling piglets Growth performance Intestinal function

1. Introduction The gut of neonatal piglet, profoundly underdeveloped and immune-deficient, is vulnerable to be damaged (Stroke et al., 2004). Therefore, the status of intestinal development of neonatal piglet has a significant influence on the growth performance and defense against pathogens during suckling period. Dietary nutritional modulation of intestinal environment has become an attractive approach to improve the gut development in neonatal piglets. Arginine is demonstrated to be necessary for the optimum growth in neonatal piglets (Wu et al., 2004). However, arginine in sow’s milk is quite deficient, thus it limits the maximal growth of piglets (Wu et al., 2004). Moreover, the endogenous arginine synthesis is decreased with the age of piglets (Wu and Knabe, 1995). To solve this paradox, researchers tried to increase arginine n

Corresponding author. Fax: þ 86 10 62733688. E-mail address: qiaoshy@mafic.ac.cn (S. Qiao).

supply to piglets through dietary arginine supplementation to lactating sows. This was not successful because of an extensive arginine catabolism in mammary glands (O’Quinn et al., 2002). On the contrary, dietary 0.2% and 0.4% arginine supplementation could dose-dependently improve the growth performance in 7- to  21-d-old piglets (Kim and Wu, 2004). However, arginine is not cost effective and has a short biological half life cycle (Wu et al., 2007a). Therefore, many researchers tried to explore alternatives for arginine. N-carbamylglutamate (NCG) is a stable analog of N-acetylglutamate (NAG), which modulates pyrroline-5-carboxylate synthase and carbamoylphosphate synthase-І, ultimately regulating intestinal endogenous citrulline and arginine synthesis (Wu et al., 2004). NCG has been demonstrated to exert comparable function like NAG (Wu et al., 2004). In rats, dietary supplementation of 0.1% NCG improved embryo implantation during early pregnancy (Zeng et al., 2012). In sows, dietary 0.1% NCG supplementation during late gestation improved placental and umbilical vascular function to support fetal development probably

http://dx.doi.org/10.1016/j.livsci.2015.09.004 1871-1413/& 2015 Published by Elsevier B.V.

Please cite this article as: Zeng, X., et al., Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.09.004i

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through providing more nutrients and oxygen from the maternal to the fetus (Yang et al., 2011; Liu et al., 2012; Wu et al., 2012a). In weaning piglets, dietary 0.08% NCG supplementation improved intestinal growth and integrity, increased the mRNA expression of intestinal amino acid transporters, affected intestinal mucosal morphology and epithelial cell proliferation (Wu et al., 2010, 2012b; Yang et al., 2013). In neonatal piglets, oral administration of 50 mg of NCG/kg body weight twice daily increased the protein synthesis in skeletal muscle (Frank et al., 2007). According to Zhang et al. (2013), 50 mg NCG/kg body weight supplementation to milk replacer improved intestinal mucosal immunity in Escherichia coli-challenged neonatal piglets. However, few studies have analyzed the effects of NCG supplementation on intestinal digestive enzymes, microbiota population, and immunity under physiological condition. Thus, the objective of this study was to investigate the effects of oral NCG administration from d 1 to d 14 after birth on intestinal digestive enzymes, microbiota numbers, and immunity under physiological condition in neonatal piglets, ultimately finding a replacement for arginine.

2. Materials and methods 2.1. Animals, experimental design, and diets All animals used in this study were housed and handled according to the established guidelines of the China Department of Agriculture. All procedures used in this experiment were approved by the China Agricultural University Animal Care and Use Committee. One-d old newborn piglets (Landrace  Yorkshire) from sows of the same parity order (parity 1, sows’ information were shown in Supplementary Table S1) were used in this study (n ¼48 piglets, 4 L). All the piglets were sow-reared. The litter size was averaged to 12 piglets per sow, with cross-fostering done within 24 h after birth. The average initial body weight was 1.57 70.04 kg. The piglets were allotted to the following 4 treatments (12 piglets/ treatment): (1) 0.52 g/kg body weight (BW) of L-Alanine (alanine, control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW BW NCG (100 mg NCG). NCG was dissolved in saline. L-Alanine and L-Arginine were sourced from Ajinomoto (Ajinomoto, Tokyo, Japan) and NCG was sourced from Sigma-Aldrich (Sigma-Aldrich, St. Louis, USA). L-arginine was used as a positive control because a previous study demonstrated that arginine improved the growth performance of suckling piglets. L-Alanine was used to make the iso-nitrogenous diets. The oral administration was given twice daily by using a 10 ml syringe without the needle at 0800 and 1600 from d 1 to d 14 after birth. Body weight was recorded on the morning of d 1, d 7 and d 14. Six piglets/treatment at d 7 and six piglets/treatment at d 14 were randomly selected to collect blood samples. Briefly, jugular venous blood samples were harvested and then centrifuged at 3000  g for 10 min at 4 °C to collect serum samples, which were stored at  80 °C. After blood sample collection, pigs were anesthetized with zoletil (Virbac, Carros, France) and then killed to get jejunal/ileal mucosa, and ileal samples, as well as cecal digesta. Briefly, mucosa from middle jejunum and ileum tissues was scraped with a glass slide and stored at  80 °C. Ileal samples were collected and fixed in 4% paraformaldehyde. The digesta from the middle cecum was harvested aseptically and stored at  80 °C. During the experimental period, all piglets had free access to sow’s colostrum/milk and water. During this experimental period, all piglets were free access to sow’s colostrum/milk, creep feed and water.

2.2. Analysis of serum concentrations of amino acids and immunoglobulins Concentration of serum amino acids was analyzed using a S-433D Amino Acid Analyzer (Sykam GmbH, Kleinostheim, Germany). Briefly, frozen serum samples were first thawed on ice and deproteinized using 3 mL of 10% salicylsulfonic acid/mL of serum. After a 15 min’ ice bath, the pH value for the reaction system was adjusted by adding lithium hydroxide solution (2 mol/L) and then centrifuged at 11, 000  g for 30 min at 4 °C (Beckman Optima L80-XP, Beckman Coulter Inc., Fullerton, CA). Supernatant was collected and then passed through a 0.1 μm filter. Then the supernatant was loaded to the Amino Acid Analyzer. Assays for serum concentration of immunoglobulins (IgA, IgG, IgM) were performed using specific swine ELISA Ig quantification kits (Cusabio Biotech Company, Wuhan, China), based on the procedures of the manufacture’s instruction. The assay procedures for all the three kits were similar. Briefly, (1) Set a Blank well without any solution. (2) Add 50 μl of Standard or Sample per well. Standard need test in duplicate. (3) Add 50 μl of HRP-conjugate to each well immediately (not to Blank well). (4) Mix well and then incubate for 40 min at 37 °C. (5) Aspirate each well and wash, repeating the process four times for a total of five washes. (6) Add 90 μl of TMB Substrate to each well. Incubate for 20 min at 37 °C. Protect from light. (7) Add 50 μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing. (8) Determine the optical density of each well within 5 min, using a microplate reader set to 450 nm. The R2 values of the linear regression equations for IgA, IgG, and IgM were 0.9938, 0.9928, and 0.9941 respectively. The linear regression lines for IgA, IgG, and IgM were shown in Supplementary Figs. S1, S2 and S3. 2.3. Immunohistochemistry The ileal samples were fixed with 4% paraformaldehyde and later embedded in paraffin. Consecutive sections (5 μm) were obtained from paraffin-fixed intestinal segments and located onto glass slides. Endogenous hydrogen peroxide was blocked by 3% hydrogen peroxide solution for 20 min at room temperature. Then, the slides were heated for 10 min in citrate buffer solution (pH 6.0) and then blocked for 20 min with PBS containing 10% goat serum. The slides were incubated with anti-CD4, anti-CD8 or anti-CD19 primary antibodies (rabbit polyclone antibodies against human), (ZSGB-BIO, Beijing, China) at a ratio of 1:200 overnight at 4 °C. After being washed with PBS, the slides were incubated with peroxidase conjugated anti-IgG antibody (1:500 in PBS, v/v) for 30 min at 37 °C. Slides were exposed in DAB substrate reagent (ZSGB-BIO, Beijing, China) and counterstained with Mayer’s hematoxylin (ZSGB-BIO, Beijing, China). For the negative control, the antibodies were substituted by PBS. Immunochemical staining was photographed by using the Leica DFC 320 digital camera (Leica Microsystems). The optical density was obtained by computerassisted image analysis (Image-Pro Plus; Media Cybernetics). Eight microscopic fields for each section were randomly selected to be quantified. 2.4. Analysis of cytokines in ileal homogenates and secretory immunoglobulin A (sIgA) in ileal mucosa Ileal samples of 1.0 g were weighed and added with 10 mL phosphate buffered saline, and were homogenized for 60 s on ice and then centrifuged at 11,000  g for 15 min at 4 °C. The supernatants were stored at 20 °C until analysis. Concentrations of cytokines (IL-1 and IL-6) in ileum were measured using ELISA kits (Cusabio Biotech Company, Wuhan, China). The assay procedures

Please cite this article as: Zeng, X., et al., Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.09.004i

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for IL-1 and IL-6 kits were as follows. (1) Add 100 μl of standard and sample per well. Cover with the adhesive strip provided. Incubate for 2 h at 37 °C. 4. (2) Remove the liquid of each well, donot wash. (3) Add 100 μl of Biotin-antibody (1  ) to each well. Cover with a new adhesive strip. Incubate for 1 h at 37 °C. (4) Aspirate each well and wash, repeating the process two times for a total of three washes. Wash by filling each well with 200 μl Wash Buffer, and let it stand for 2 min. After the last wash, remove any remaining wash Buffer by aspirating ordecanting. Invert the plate and blot it against clean paper towels. (5) Add 100 μl of HRP-avidin to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 h at 37 °C. (6) Repeat the aspiration/wash process for five times as in step 4. (7) Add 90 μl of TMB Substrate to each well. Incubate for 15–30 min at 37 °C. Protect from light. (8) Add 50 μl of Stop Solution to each well, gently tap the plate to ensure thorough mixing. (9) Determine the optical density of each well within 5 min, using a microplate reader set to 450 nm. The R2 values of linear regression equations for IL-1 and IL-6 were 0.9924 and 0.9992. The linear regression lines for IL-1 and IL-6 were shown in Supplementary Figs. S4 and S5. After the digesta was removed from the ileal samples, the mucosa was gently scraped with a glass slide and then stored at 80 °C until use. Mucosal samples of 0.1 g were homogenized in 5 mL PBS with 1% protease inhibitor cocktail (Applygene, Beijing, China) for 60 s on ice. The homogenates were centrifuged at 11,000  g for 15 min at 4 °C. The sIgA concentrations in the supernatant were measured using a swine ELISA kit (Cusabio Biotech Company, Wuhan, China), and were normalized for the weight of the ileal sample. The assay procedure was indicated above. The R2 value of linear regression equations for sIgA was 0.994. The linear regression lines for sIgA were shown in Supplementary Fig. S6.

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(3) The absorbance was measured at 505 nm. The activities were expressed as U per mg protein. 2.6. Cecum microbiota quantification In vitro survival of lactobacillus spp., E. coli, and total anaerobes were determined according to the methods of Guo et al. (2006) and Mikkelsen et al. (2003) with certain modifications. In brief, before enumeration, frozen digesta samples were incubated at 4 °C for 1 h. Thereafter, 0.1 g of digesta was taken from each sample and serially diluted from 10  1 to 10  6 for enumeration in sterile physiological saline. E. coli was cultured on MacConkey agar (Beijing Aoboxing Universeen Bio-Tech Co., LTD., Beijing, China). Lactobacilli spp. were determined using MRS (De Man, Rogosa, Sharpe) agar (Oxoid Ltd., Hampshire, UK) and total anaerobes were cultivated on Chloramphenicol Bromophenol Blue agar (CBB, Beijing Aoboxing Bio-tech Co., LTD., China). Both the plates of lactobacilli spp. and total anaerobes were incubated inside an anaerobic jar with AnaeroPack Anaero (Mitsubishi Gas Chemical Company, Tokyo, Japan). All plates were incubated at 37 °C for 24 h. The microbial enumerations were expressed as log10 CFU/g (colony-forming units per gram). 2.7. Statistical analysis Body weight data were analyzed by PROC MIXED model of SAS with repeated measurements. Statistical differences were separated by PDIFF option of SAS. Data except body weight were analyzed by ANOVA using the General Linear Model procedures of the Statistical Analysis System (SAS Inst Inc., Cary, NC). P o0.05 was regarded as significant.

2.5. Determination of jejunal mucosal disaccharidase activities 3. Results Jejunal mucosal samples were handled as ileal mucosal samples, described above. The protein concentration in the supernatant was determined by BCA Protein Assay Kit (Pierce, Rockford, USA). Disaccharidase (sucrase, lactase and maltase) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjingjiancheng, Nanjing, China) and the activities of jejunal mucosal disaccharidases were determined according to the manufacture’s instructions. Briefly, (1) Add 10 μl supernatant and 20 μl substrate together and vortex thoroughly, then incubate in water bath at 37 °C for 20 min. (2) Add 10 μl of terminating reagent and 1000 μl of chromogenic agent to the above mixture and vortex thoroughly, then incubate in water bath at 37 °C for 15 min.

3.1. Growth performance There was no effect of treatments on average daily gain of piglets from d 1 to d 7 after birth (Table 1, P 40.1). During d 7 to d 14 after birth, piglets supplemented with 50 mg/kg BW NCG tended to increase the average daily gain compared with those supplemented with the alanine (control) (Table 1, P¼ 0.08), while significantly increased average daily gain compared with those supplemented with 100 mg/kg BW NCG (Table 1, P¼ 0.05). Piglets supplemented with 50 mg/kg BW NCG had increased average daily gain during days 1–14 after birth compared with those

Table 1 Growth performance of the piglets1. Items

BW (kg) d1 d7 d 14 ADG (g) d 1–7 d 7–14 d 1–14

Crt

Arg

NCG (mg/kg BW) 50

100

SEM

Comparison P value Ctr:Arg

Crt:NCG50

Crt:NCG100

Arg:NCG50

Arg:NCG100

NCG50:NCG100

1.57 2.68 4.5

1.58 2.72 4.55

1.57 2.73 4.69

1.57 2.69 4.42

0.04 0.05 0.05

0.98 0.60 0.60

0.98 0.85 0.22

0.95 0.54 0.67

0.96 0.75 0.09

0.94 0.92 0.35

0.98 0.68 0.43

158 259 210

163 253 210

167 267 220

160 252 206

4.79 5.53 3.66

0.52 0.45 0.88

0.21 0.33 0.05

0.84 0.34 0.56

0.55 0.08 0.07

0.66 0.85 0.66

0.30 0.05 0.03

a, b

Means within a row with different superscripts are significantly different (Po 0.05).

1 4 treatments were as follows: (1) 0.52 g/kg body weight (BW) of L-Alanine (control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW NCG (100 mg NCG). Six piglets from each treatment were randomly selected to be killed at d 7, with the remaining killed at d 14 (at d 1, 7 and 14, n ¼12, 12 and 6, respectively). Crt: control; Arg: arginine

Please cite this article as: Zeng, X., et al., Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.09.004i

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Table 2 Concentrations of serum amino acids in suckling piglets (μmol/L)1. Items

d7

SEM

Control

Arginine

Arginine Histidine Lysine Alanine Glutamine Glutamate Citrulline Ornithine

74.2 82.2 230.2 832.5a 605.2a 161.6a 78.3a 68.7a

b

95.7 83.5 232.4 825.6b 622.8b 182.0a 90.5b 81.4b

d 14

NCG(mg/kg BW) 50

a

P value

Control

Arginine

115.1 84.1 233.1 824.4b 604.7a 261.6b 88.9b 83.7b

50 c

116.6 83.2 2332 823.9b 603.3a 263.1b 85.7b 80.5b

6.4 1.11 3.26 0.55 0.52 24.8 0.25 0.23

a

o0.01 0.46 0.57 o0.01 o0.01 0.01 o0.01 o0.01

b

156.2 80 235.4 776.6 577.8a 201.0a 65.7a 85.2a

P value

0.45 0.65 2.2 0.60 0.52 18.06 0.40 3.6

0.03 0.1 0.81 0.36 o 0.01 o 0.01 o 0.01 o 0.05

NCG(mg/kg BW)

100 c

SEM

100 c

180.2 79.4 234.1 778.4 561.9b 235.0ab 79.4b 100.4b

191.6c 80.1 234.2 782.8 561.7b 267.0bc 74.3b 102.9b

190.3 81.7 235.7 783.2 564.2b 304.0c 76.5b 101.5b

a, b, c

Means within a row with different superscripts on the same day are significantly different (n ¼6, Po 0.05).

1

4 treatments were as follows: (1) 0.52 g/kg body weight (BW) of L-Alanine (control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW NCG (100 mg NCG). Six piglets from each treatment were randomly selected to be killed at d 7 and d 14 for harvesting blood samples.

supplemented with alanine (Table 1, P ¼0.05) and 100 mg/kg BW NCG (Table 1, Po0.05), while tended to increase the average daily gain compared with those supplemented with arginine (Table 1, P ¼0.07).

Table 3 Concentrations of serum Immunoglobulins (μg/mL) in different treatments at d 7 and 141. Items

Control

Arginine

3.2. Concentrations of serum amino acids At d 7 of the experiment, the concentration of serum arginine in the two NCG groups was increased compared with that in the control and the arginine group (Table 2, Po 0.05). The concentration of serum glutamate, ornithine and citrulline in the two NCG groups was also increased markedly, compared with that in the control group (Table 2, P o0.05). Serum glutamine concentration was not increased in the two NCG groups, compared with the control group nevertheless it was lower (Table 2, P o0.05). At d 14 of the experiment, serum arginine concentration in the two NCG groups was increased compared that in the control group and arginine group (Table 2, P o0.05). The concentration of serum glutamate, citrulline and ornithine was increased in the two NCG groups compared with the control group (Table 2, P o0.05). Serum glutamine concentration in the arginine and two NCG groups was decreased compared with that in the control group (Table 2, P o0.05). 3.3. Serum concentration of immunoglobulins There was no effect of supplementation on piglet serum IgA concentration on d 7 (P 40.10, Table 3). However, oral administration of 50 mg/kg BW NCG increased serum IgA levels at d 14 (Table 3, P o0.05). There was no effect of supplementation on serum IgG and IgM concentration at d 7 and d 14 (P4 0.10). 3.4. Ileal cytokines, sIgA, and percentage of CD4 þ , CD8 þ and CD19 þ lymphocytes at d 14 There was no effect of NCG supplementation on concentration of ileal IL-1 and IL-6 (Table 4), and percentage of CD4 þ , CD8 þ and CD19 þ lymphocytes (Table 5, P 40.10). However, concentrations of ileal sIgA tended to be increased in the ileum of piglets supplemented with arginine or 50 mg/kg BW NCG (Table 4, P¼ 0.08). 3.5. Jejunal mucosal disaccharidase activities At d 7, the activity of lactase in 50 mg NCG group was increased compared with the other treatments (Table 6, P o0.05). However,

IgG 7d 14 d IgA 7d 14 d IgM 7d 14 d

NCG (mg/kg BW) 50

100

SEM

P value

9478 6631

8826 6429

8690 6812

8206 5826

532 360

0.39 0.67

457 114a

477 126b

439 150b

410 110a

28 8

0.12 0.05

497 476

538 548

520 515

544 527

26 22

0.35 0.40

a,b

Means within a row with different superscripts on the same day significantly differ (n¼ 6, P o0.05).

1 4 treatments were as follows: (1) 0.52 g/kg body weight (BW) of L-Alanine (control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/ kg BW NCG (100 mg NCG). Six piglets from each treatment were randomly selected to be killed at d 7 and d 14 for harvesting blood samples.

Table 4 Cytokines and sIgA concentrations of ileum homogenates in different treatments at d 14 (n¼ 6)1. Items

Control Arginine NCG(mg/kg BW) 50

IL-6 (pg/mL homogenates) IL-1 (pg/mL homogenates) sIgA (μg/g)

SEM P value

100

2.7

1.9

2.1

2.6

0.3

0.69

6.0

4.1

4.1

4.9

0.7

0.46

38.2

41.8

43.8

39.1

1.8

0.08

1 4 treatments were as follows: (1) 0.52 g/kg body weight (BW) of L-Alanine (control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW NCG (100 mg NCG). The remaining six piglets from each treatment were killed at d 14 for harvesting the middle ileum samples.

there was no effect of supplementation on the activity of maltase and sucrase (P 40.10, Table 6). At d 14, there was no significant difference in activities of the three disaccharidases (Table 6).

Please cite this article as: Zeng, X., et al., Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.09.004i

X. Zeng et al. / Livestock Science ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 5 Numbers of ileal lamina propria lymphocyte subsets in piglets at d 14 (n ¼6)1. Items

CD4 þ CD8 þ CD19 þ

Control

22 20 30

Arginine

28 20 34

NCG(mg/kg BW) 50

100

25 21 35

25 18 31

SEM

P value

1.35 0.98 1.50

0.32 0.90 0.71

1 4 treatments were as follows: (1) 0.52 g/kg body weight (BW) of L-Alanine (control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW NCG (100 mg NCG). The remaining six piglets from each treatment were killed at d 14 for harvesting the middle ileum samples.

3.6. Cecal microbial number At d 7, the number of E. coli, lactobacillus spp. and anaerobic bacteria was not affected by treatments. At d 14, the number of E. coli was still not different among treatments. However, the number of lactobacillus spp. in arginine treatment was lower than the two NCG treatments (Table 7, P o0.05). The total number of anaerobic bacteria in the control was lower than that in the other three treatments (Table 7, P o0.05).

4. Discussion Previous study indicated that piglets received 50 mg of NCG/kg body weight every 12 h for 7 d gained much more weight than the control pigs (Frank et al., 2007). In this study, oral administration of 50 mg of NCG/kg body weight twice daily for two weeks could significantly increase the average daily gain in suckling piglets. Moreover, serum concentrations of arginine and arginine family of amino acids like glutemate, citrulline, and ornithine were also significantly increased by oral NCG administration, which further verified the increase in endogenous synthesis of arginine and arginine family of amino acids. Therefore, it was speculated that the improvement in average daily gain induced by oral administration of NCG in the present study probably was associated with the increase in intestinal endogenous synthesis of arginine and arginine family of amino acids in piglets, which supplied sufficient arginine and arginine family of amino acids for growth. Additionally, arginine family of amino acids act as important precursors of protein and nucleic acid synthesis, and also act as signaling molecules, to support gastrointestinal functions (Wu et al., 2007b). Unexpectedly, in this study, dietary arginine supplementation did not improve the growth of piglets. The possible reasons might be associated with the different breeds (offspring of PIC Cambrough-22  PIC versus offspring of Landrace  Yorkshire) and ages of piglets (1-d-old versus 7-d-old), as well as the living

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environment (university swine research center versus pig farm). Nutrient digestion directly influences the growth rate in animals. Therefore, the activities of intestinal digestive enzymes are important factors to evaluate the capability of nutrient digestion. Studies have demonstrated that some amino acids and their metabolites could enhance the activities of some digestive enzymes (Shimizu et al., 1993; Harada et al., 1994; Xu et al., 2012). For example, oral spermine administration dose-dependently increased intestinal maltase, sucrase, and lactase activities in suckling rats (Shimizu et al., 1993; Harada et al., 1994). A mixture of dietary arginine and glutamine supplementation increased the sucrase and maltase activities in weaned piglets (Xu et al., 2012). In the present study, dietary NCG supplementation increased lactase activity in suckling piglets at d 7, indicating a beneficial effect on lactose digestion. Another interesting result in this study was that the total number of cecal lactobacillus and anaerobic bacteria in the NCG treated groups was significantly higher than that in the control group. This was probably because lactobacillus and anaerobic bacteria in the cecum could utilize arginine, which might improve the growth of these bacteria. Arginine, glutamate, and several other amino acids were demonstrated to be largely utilized by the bacteria of small intestines in pigs (Dai et al., 2010, 2012). Amino acids, including essential and non-essential amino acids, are proved to be metabolized in the gastrointestinal tract, which plays important roles in maintaining gut health and functions, including balance of gut microflora (Dai et al., 2010). Therefore, we could speculate that dietary NCG supplementation increased the synthesis and utilization of arginine, which improved the growth of cecal lactobacillus and anaerobic bacteria in neonatal piglets. This was beneficial for maintaining gastrointestinal function and health in neonatal piglets. Adequate supply of immunoglobulin plays a critical role in changing the situation of the low viability and high mortality of neonatal piglets in swine farm (Dividich et al., 2005; Bikker et al., 2010). The intake of colostrum, the main source of immunoglobulin, could be very limited in low birth weight piglets (Bikker et al., 2010). Besides, the content of the immunoglobulin in colostrums was decreased dramatically after 36 h after birth and could be hardly absorbed by the piglets (Klobasa et al., 1986). Therefore it is a very effective strategy to increase the immunoglobulin synthesis by dietary supplementation of some nutrient sources which might induce the immunoglobulin synthesis in piglets to enhance the ability of disease resistance. In this study, our data indicated that 50 mg/kg BW NCG supplementation significantly increased the concentrations of serum IgA and tended to increase the concentration of intestinal sIgA, which was confirmed with previous study that there was a strong positive correlation between serum IgA concentration and local intestinal production (Vaerman et al., 1997). As intestinal sIgA is the first line of

Table 6 Activities of jejunal mucosal disaccharidases of the suckling piglets at d 7 and d 14 (U/mg protein)1. Items

d7 Control

Maltase Sucrase Lactase

5.48 6.81 69.11a

SEM Arginine

5.78 7.05 69.00a

P value

NCG(mg/kg BW) 50

100

6.15 6.88 79.62b

6.08 7.08 68.70a

d 14 Control

0.88 0.86 2.41

0.95 1.00 0.01

7.12 7.85 72.50

Arginine

7.23 7.64 74.26

SEM

P value

1.09 0.89 9.11

0.88 0.99 0.79

NCG(mg/kg BW) 50

100

7.05 8.05 85.00

8.16 7.69 73.00

a, b

Means within a row with different superscripts on the same day are significantly different (n¼ 6, Po 0.05).

1 4 treatments were as follows: (1) 0.52 g/kg body weight (BW) of L-Alanine (control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW NCG (100 mg NCG). Six piglets from each treatment were randomly selected to be killed at d 7 and d 14 for harvesting jejunal mucosal samples.

Please cite this article as: Zeng, X., et al., Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.09.004i

X. Zeng et al. / Livestock Science ∎ (∎∎∎∎) ∎∎∎–∎∎∎

6

Table 7 The microbial number in the cecum of the suckling piglets at d 7 and d 14 (log10CFU/g)1. Microorganisms

d7 Control

Escherichia coli Lactobacillus spp. Total anaerobes

4.57 5.93 5.02

SEM Arginine

4.81 6.32 5.93

P value

NCG(mg/kg BW) 50

100

4.91 6.29 5.16

5.47 6.28 5.36

d 14 Control

0.51 0.33 0.42

0.64 0.82 0.46

4.74 5.78bc 4.75b

Arginine

4.69 5.57c 6.09a

SEM

P value

0.52 0.27 0.28

0.77 o0.01 o0.01

NCG(mg/kg BW) 50

100

4.61 6.48ab 6.06a

5.12 6.74a 5.92a

a, b, c

Means within a row with different superscripts on the same day are significantly different (n ¼6, Po 0.05).

1

4 treatments were as follows: (1) 0.52 g/kg body weight (BW) of L-Alanine (control); (2) 0.31 g/kg BW L-Arginine
HCl (arginine); (3) 0.52 g/kg BW L-Alanine plus 50 mg/kg BW NCG (50 mg NCG) and (4) 0.52 g/kg BW L-Alanine plus 100 mg/kg BW NCG (100 mg NCG). Six piglets from each treatment were randomly selected to be killed at d 7 and d 14 for harvesting cecal digesta samples.

intestinal barrier, the decrease in intestinal sIgA would probably increase the possibility of infection by pathogens in neonatal piglets. As the synthesis of IgA is associated with the numbers of lymphocytes and cytokines, therefore the number of CD4 þ , CD8 þ , and CD19 þ cells in lamina propria and concentrations of IL-1, IL-6 in ileal homogenates were detected. Our data indicated that dietary NCG supplementation had no significant effects on the numbers of lymphocytes or the concentrations of cytokines. However, previous studies demonstrated that dietary arginine supplementation could enhance the proliferation of lymphocytes and modulate the concentrations of cytokines (Kobayashi et al., 1998; Ochoa et al., 2001). Our previous data also indicated that dietary NCG supplementation significantly increased the proliferation of CD4 þ cells and trended to increase CD19 þ cells and promoted IL-10 production in neonatal piglets after E coli. Challenge (Zhang et al., 2013). The reason for this discrepancy may be that under physiological condition, the lymphocytes are not so sensitive to dietary NCG supplementation, compared with that under pathological condition.

5. Conclusion In conclusion, oral NCG administration improved suckling piglets’ growth performance, increased endogenous synthesis of arginine and arginine family of amino acids, jejunal lactase activity, cecal Lactobacillus spp. and anaerobic bacteria population, and secretion of ileal sIgA. These findings have important implications in enhancing the gastrointestinal health status and growth performance in suckling piglets.

Conflict of interest The authors declare that there is no conflict of interest.

Acknowledgments This study was supported by the Science Foundation for the Excellent Youth Scholars of Ministry of Education of China (No. 2014JD019), the National Natural Science Foundation of the PR of China (No. 31301980), and National Key Basic Research Program (2012CB124704). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.livsci.2015.09.004.

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Please cite this article as: Zeng, X., et al., Oral administration of N-carbamylglutamate might improve growth performance and intestinal function of suckling piglets. Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.09.004i