Key words: intestinal inflammation, piglets, spray-dried plasma powder, yeast cell wall

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1 Effect of replacing spray-dried plasma powder with yeast cell wall in creep feed on intestinal inflammatory status of piglets during the suckling and nursery periods 1 H. J. Ni,* 2 Y. Z. Zhang,* 2 J. Li,* P. Wang,* L. Chen,* # X. Y. Bao,* # Y. Y Zhou, Z. M. Feng,* 3 and Y. L. Yin* 3 *Key Laboratory for Agro-Ecological Processes in Subtropical Region, Hunan Research Center of Livestock and Poultry Sciences, South Central Experimental Station of Animal Nutrition and Feed Science in the Ministry of Agriculture, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Hunan, China; Hinapharm Pharmaceutical Co., Ltd, Foshan, Guangdong, China; University of Chinese Academy of Sciences, Beijing, China; Veterinary Faculty, Hunan Agricultural University, Changsha, Hunan, China; # College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan, China; and Hunan Co-Innovation Center of Animal Production Safety, Changsha, Hunan, China ABSTRACT: In the present study, the effect of replacing spray-dried plasma powder (SDPP) with yeast cell wall (YCW) in creep feed on the intestinal inflammatory status of piglets was evaluated. Five iso-nitrogenous and iso-caloric diets were supplied to piglets: 1) standard diet (BD), 2) 1% YCW+3% SDPP (LY), 3) 2% YCW+2% SDPP (MY), 4) 4% YCW (HY), and 5) 4% SDPP (ZY). The results showed that feeding the LY diet had a better effect on improving duodenal and ileal villous height of piglets during suckling and nursery periods compared with the ZY group (P < 0.05). Besides, feeding YCW (LY, MY, and HY) increased mrna expression of Toll-like receptor 4 (TLR4) in the duodenum and jejunum compared with the BD group (P < 0.05). Also, the HY diet up-regulated the mrna expression of nucleotide-binding oligomerization domain 2 (NOD2) in the ileum compared with the ZY diet (P < 0.05). Furthermore, the HY diet increased the mrna expression of IL-6 in the duodenum and ileum compared with the BD group. These results suggest that YCW had a benefit in enhancing intestinal immune defenses. Collectively, the current results indicate that YCW can be used as a substitute for SDPP in creep feed for its beneficial effects on improving intestinal villous height and promoting intestinal immune defenses. Key words: intestinal inflammation, piglets, spray-dried plasma powder, yeast cell wall 2016 American Society of Animal Science. All rights reserved. J. Anim. Sci :95 99 doi: /jas INTRODUCTION Various factors induce stress in piglets during the weaning period, which seriously affects their growth and development and can induce diseases or even death (Campbell et al., 2013). Spray-dried plasma 1 The present study was jointly supported by the Project of Cooperation Between Schools and Enterprises supported by Hinapharm Pharmaceutical Co., Ltd (Y ), the National 948 Project from the Ministry of Agriculture of China (2015Z74), and funds from the Ministry of Agricultural Science and Technology (2014GB2D200216). 2 These authors contributed equally to the paper. 3 Corresponding authors: zemengfeng2006@163.com; yinyulong@ isa.ac.cn 95 powder (SDPP) is a feedstuff made from animal blood and is frequently used in creep feed. However, it is expensive and variable in quality (Zhang et al., 2015). As a novel protein source, yeast cell wall (YCW) provides not only large content of protein but also many nutrition components, such as β-glucan, mannan-oligosaccharide, and glycoprotein (Aguilar- Uscanga and François, 2003), which are a benefit in promoting feed intake and immunity (Hooge, 2003; Gómez-Verduzco et al., 2009). Meanwhile, several studies demonstrated that YCW may have the ability to retard stress similar to SDPP but with manageable quality (Santin et al., 2001; Aguilar-Uscanga and François, 2003), which makes it a possible substitute candidate for SDPP. Thus, here we studied the effect of YCW as a substitute for SDPP in creep feed on

2 96 Ni et al. Table 1. Ingredients and nutrient composition of diets BD LY MY HY ZY Ingredient composition (%) Extruded soybean Fermented soybean Soybean meal W hey powder CP2%, Lactose 80% Extruded corn Corn Soybean oil Mountain flour Monocalcium phosphate Fish meal YCW SDPP Glucose Lys (98%) dl-met l-thr l-try Sucrose Premix Calculated analysis CP % DE, kcal/kg 3, , , , , Premix (%): anhydrous citric acid: 60.24; choline chloride (50%): 4.02; ZnO: 12.05; antimold agent: 2.00; antioxidants: 1.20; vitamins: 1.61; organic minerals: 6.02; colistin sulfate (10%): 0.80; olaquindox (50%): 0.80; 10% hygromix (hinapharm Pharmaceutical Co., Ltd): 1.2; antibacterial peptide (Hinapharm Pharmaceutical Co., Ltd): 1.2; flavoring agent (fragrant and sweet): 4.02; sweetener: 0.40; high temperature resistant phytase (5,000 U): 0.40; enzymes: 0.80; vitamin C: 1.20; rice bran meal (15% CP): the feed intake and intestinal inflammatory status of piglets during the suckling and nursery period. MATERIALS AND METHODS Animals and Treatments Initially, 25 healthy pregnant sows with similar growth performance and parities were selected. Then the litter size was averaged to 10 piglets per sow, with cross-fostering done at 7 d postpartum. Meanwhile, the litters were randomly assigned into 5 groups, and thus, 250 piglets were employed. Five iso-nitrogenous and iso-caloric diets (Table 1) were used in this study: 1) standard diet (BD), 2) 1% YCW+3% SDPP (LY), 3) 2% YCW+2% SDPP (MY), 4) 4% YCW (HY), and 5) 4% SDPP (ZY). All the piglets were weaned at 18 d old, and one piglet was randomly selected from each litter and sacrificed by jugular puncture under general anesthesia via the intravenous injection of 4% sodium pentobarbital solution (40 mg/kg BW), and the intestinal samples were immediately collected. The rest of the piglets were fed with the diets as previously assigned until 25 d old, when one piglet was randomly selected from each litter and sacrificed along with sample collection. All experimental procedures used in this study were approved by the Animal Care and Use Committee of the Chinese Academy of Sciences. Intestinal Morphology Intestinal samples from piglets including the duodenum, jejunum, and ileum were collected and embedded in paraffin. Cross sections of the segments were cut and stained with hematoxylin and eosin, and the villus height and crypt depth were measured according to a previous study (Yin et al., 2015). Analysis of Intestinal Immune-Related Genes of Nursery Piglets by Real-Time PCR Immune-related genes expressed in the duodenal, jejunal, and ileal tissues were detected by relative quantitative real-time PCR. The procedures for the detection of nucleotide-binding oligomerization domain 2 (NOD2), double-stranded RNA-dependent protein kinase (PKR), trefoil factor family (TFF), eukaryotic translation initiation factor 5a (eif-5a), Toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), IL-1β,

3 Yeast cell wall for piglets 97 Table 2. Effects of substitute spray dried plasma powder (SDPP) for yeast cell wall (YCW) in creep feed on the intestinal morphology of piglets, n = 5 BD LY MY HY ZY Suckling period (μm) Duodenum VH ± 40 a 499 ± 39 c 490 ± 40 bc 489 ± 26 bc 482 ± 39 b CD ± ± ± ± ± 38 Jejunum VH ± 25 a 460 ± 28 c 452 ± 14 b 455 ± 26 c 449 ± 17 b CD ± ± ± ± ± 20 Ileum VH ± 29 a 376 ± 35 c 368 ± 29 c 365 ± 45 bc 358 ± 42 b CD ± ± ± ± ± 28 Nursery period (μm) Duodenum VH ± 32 a 485 ± 28 c 480 ± 34 bc 486 ± 24 bc 479 ± 35 b CD ± ± ± ± ± 26 Jejunum VH ± 15 a 443 ± 26 b 449 ± 31 ab 453 ± 27 b 447 ± 19 ab CD ± ± ± ± ± 21 Ileum VH ± 31 a 369 ± 38 b 365 ± 42 b 364 ± 13 ab 360 ± 52 ab CD ± ± ± ± ± 36 a c Values in the same row with different superscript letters are significantly different (P < 0.05). 1 VH = villous height. 2 CD = crypt depth. IL-6, IL-8, and IL-10 were designed and performed according to previous reports (Tohno et al., 2008; Gao et al., 2012; Feng et al., 2014). The results are presented as fold changes using the 2 ΔΔCT method. Statistical Analysis First, the tests for normal distribution (Shapiro- Wilk test) and equal variance were performed. If both tests were passed, the data were analyzed by a one-way analysis of variance followed (the Student-Newman- Keuls test) using SPSS Differences were considered to be significant at P < 0.05 (Wu et al., 2012). RESULTS AND DISCUSSION The intestinal morphology is an important index to evaluate the effects of YCW. Consistent with previous research (Shen et al., 2009), YCW had a positive effect on improving the intestinal villus height of piglets. The results (Table 2) also suggested that feeding the LY diet had a better effect on improving duodenal and ileal villous height of piglets during the suckling and nursery period compared with the ZY group (P < 0.05). This was probably because YCW contains abundant β-glucan and oligosaccharides, which were considered helpful in enhancing immunity and protection against pathogen infection (Hooge, 2003; Stuyven et al., 2009) The principal role of TLR signaling in the intestine is defending against pathogens, and TLR4, like other TLR, plays an important role in protection against enteric pathogens, while NOD2 is an additional pattern recognition receptor that is required for defense against invasive enteric pathogens (Abreu et al., 2005). Our results (Fig. 1) showed that feeding YCW (LY, MY, and HY diets) increased the mrna expression of TLR4 in the duodenum and jejunum compared with the BD group (P < 0.05). Also, feeding the HY diet up-regulated the mrna expression of NOD2 in the ileum compared with the ZY group (P < 0.05). Furthermore, the HY diet increased mrna expression of IL-6 in the duodenum and ileum compared with the BD group, which was important in regulating innate adaptive immunity (Martinez et al., 2013). These results suggest that YCW helped promote the intestinal immune response to infections and enhance intestinal immune defenses. In conclusion, feeding YCW had beneficial effects on improving intestinal villous height and promoting intestinal immune defense. Thus, YCW can be used as a substitute for SDPP in creep feed. LITERATURE CITED Abreu, M. T., M. Fukata, and M. Arditi TLR signaling in the gut in health and disease. J. Immunol. 174: doi: /jimmunol Aguilar-Uscanga, B., and J. M. François A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Lett. Appl. Microbiol. 37: doi: /j x x Campbell, J. M., J. D. Crenshaw, and J. Polo The biological stress of early weaned piglets. J. Anim. Sci. Biotechnol. 4:19. doi: / Feng, Z., T. Li, C. Wu, L. Tao, F. Blachier, and Y. Yin Monosodium l-glutamate and dietary fat exert opposite effects on the proximal and distal intestinal health in growing pigs. Appl. Physiol. Nutr. Metab. 40: doi: /apnm

4 98 Ni et al. Figure 1. The effect of replacing spray dried plasma powder (SDPP) with yeast cell wall (YCW) in creep feed on the mrna expressions of immunerelated genes in (A) duodenum, (B) jejunum, and (c) ileum of nursery piglets, n = 5. a c Values with different superscript letters are significantly different (P < 0.05).

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