Inclusion of linseed and linseed expeller meal in piglet diets affects intestinal gene expression profiles

Livestock Science 108 (2007) 23 – 25 www.elsevier.com/locate/livsci

Inclusion of linseed and linseed expeller meal in piglet diets affects intestinal gene expression profiles☆ A.J.M. Jansman ⁎, T.A. Niewold 1 , M.M. Hulst Animal Sciences Group, P.O. Box 65, 8200 AB Lelystad, The Netherlands

Abstract Linseed as a feed ingredient contains a number of constituents, such as mucilage, gel forming polysaccharides, structural carbohydrates and ω-3 fatty acids, which could have functional properties in relation to maintaining and supporting gastrointestinal health in post weaning piglets. Intestinal gene expression was studied in weanling piglets fed diets containing either or not linseed or linseed expeller meal. Piglets were either or not orally challenged with an enterotoxigenic strain of E. coli K88. Pairwise comparisons identified several mRNAs expressed significantly different, in particular between treatments C− (control, not challenged) and LS− (linseed, not challenged) and LS+ (linseed, challenged) and LSM+ (linseed expeller meal, challenged). The intestinal micro-array technique is a promising tool to study the relationship between nutrition and feeding and gut tissue responses under defined conditions. © 2007 Elsevier B.V. All rights reserved. Keywords: Linseed; Weaning; Intestinal gene expression; Piglets

1. Introduction The recent ban on the use of growth promoting antibiotics in animal feeds has put pressure on the development of new feeding strategies and formulations to support performance and gut health. Especially, piglets in the period after weaning are sensitive to digestive disorders and suboptimal intestinal health. This has been related to a number of factors including the process of

☆ This paper is part of the special issue entitled “Digestive Physiology in Pigs” guest edited by José Adalberto Fernández, Mette Skou Hedemann, Bent Borg Jensen, Henry Jørgensen, Knud Erik Bach Knudsen and Helle Nygaard Lærke. ⁎ Corresponding author. Tel.: +31 320 237335. E-mail address: [email protected] (A.J.M. Jansman). 1 Current address: Katholieke Universiteit Leuven Kasteelpark Arenberg 30, Leuven, 3001, Belgium.

1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.013

weaning, social stress, change of environment, change of diet characteristics and low feed intake (Pluske et al., 1997). Diet composition could potentially affect the course of this transition period. Both full fat linseed (Linum usitatissimum) and fat extracted linseed expeller meal have been known for their potential functional properties related to gut health and function (Oomah, 2001). Linseed contains high levels of ω-3 fatty acids, phenolic constituents such as lignans (N500 μg/g) and mucilage, a gel forming carbohydrate to which functional characteristics have been attributed (Fedeniuk and Biliaderis, 1994). The intestine is the site where dietary constituents, nutrients, microflora and mucosal tissue interact in a very complex way. These interactions contribute to the differentiation and development of functionality of intestinal tissue and cells. Genomic techniques offer possibilities to study these complex interactions by

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A.J.M. Jansman et al. / Livestock Science 108 (2007) 23–25

Table 1 Genes showing a different degree of expression comparing treatments LS− versus C− and treatments LSM+ versus LS+ 1 ID

Ratio

mRNA

Comparison linseed diet versus control diet, no E. coli challenge (LS−/C−) pl 14 A07 darmn (c) 0.9 Pan troglodytes actin, beta (ACTB), mRNA. pl 2 G12 darms 8.2 Pancreatic thread protein (PTP), mRNA (alias PAP/REG3A/HIP) pl 4 E07 darmf 4.3 THO complex 4 (THOC4), mRNA pl 0 B02 darms 0.28 mRNA similar to hydroxysteroid (17-beta) dehydrogenase 2 Comparison linseed expeller meal diet versus linseed diet, E. coli challenge (LSM+ /LS+) pl 14 A07 darmn (c) 1.2 Pan troglodytes actin, beta (ACTB), mRNA. pl 2 D04 darms 3.8 EST homoloque to GW112 protein, mRNA (alias; olfactomedin 4) pl 2 G12 darms 3.7 Pancreatic thread protein (PTP), mRNA (alias PAP/REG3A/HIP) pl 12 E06 darmn 3.4 EST homologue to cell surface mucine expressed by epithelial cells (hCSM), mRNA pl 6 B01 darmn 3.3 Heparin binding protein (HBP15/L22), mRNA pl 2 H07 darms pl 4 F08 darmf pl 3 G06 darms

0.29 0.3 0.31

Lactase-phlorizin hydrolase-1 (LCT), mRNA, Calbindin D-9k mRNA, mRNA Glutathione S-transferase (clone K37), mRNA

Acc. number

Length (bp)

E-value

gi:57977284 gi:45430002 gi:55770863 gi:73957268

235 652 280 682

3.00E− 38 1E− 118 1E− 40 1E− 84

gi:57977284 THC2244989 gi:45430002

235 589 652 199

3.00E−38 8.1E− 36 1E− 118 1E− 11

gi:47522799

213

6E− 29

gi:37779804 gi:294215 gi:1185279

706 520 668

0 3E− 149 0

1 Messenger RNA levels in two RNA pools were compared (LS− versus C−, and LSM+ versus LS+) by dual colour hybridization using fluorescent reporter molecules Cy5 (red) and Cy3 (green). Micro-array slides were spotted with a library of expressed sequence tags (EST) prepared from jejunal mucosa (Niewold et al., 2005). For each comparison dye swaps were performed. After scanning of the slides, the created images were further processed and an intensity-dependent normalisation on all data (genes) points was performed (for details see Niewold et al., 2005). A gene was considered to be differentially expressed when the mean ratio of fluorescence intensity of Cy3 over Cy5 (or Cy5 over Cy3) was more than threefold (N3.0 or b0.33). Established nucleotide sequences of the cDNA inserts (length in bp) of library EST clones (ID) were compared with the NCBI non redundant (nr) database. The accession (acc.) number of the mRNA sequence that scored the highest degree of homology (lowest E-value) is listed. In addition, the ratio of a library clone homologue to beta-actin mRNA, (pl 14 A07 darmn (c)), which showed no significant differential expression in both comparisons, is presented.

analysing gene expression profiles of e.g. intestinal tissue (van Ommen and Stierum, 2002). The present study was carried out to evaluate the effects of the use of full fat linseed and defatted linseed expeller meal as potential functional ingredients on intestinal gene expression profiles in young piglets after weaning. 2. Material and methods The complete study comprised five treatments each fed ad libitum one of three isoenergetic and nutritionally balanced experimental diets, a control diet (C), a diet with 125 g/kg full fat linseed (LS) or a diet with 85 g/kg linseed expeller meal (LSM). The control diet was based on wheat (330 g/kg), barley (300 g/kg), soybean meal (197 g/kg), potato protein (20 g/kg), fishmeal (20 g/kg), whey powder (50 g/kg), molasses (35 g/kg) and soya oil (21 g/kg) as main ingredients. Linseed was included at the expense of wheat, soybean meal and soya oil, while linseed expeller meal at the expense of wheat and soybean meal. Groups consisted of 12 single housed piglets weaned at an age of about 4 weeks (mean BW 9.1 kg). Three of the five groups (C+, LS+, LSM+) were challenged with about 2 × 109 colony forming units of an enterotoxigenic strain of E. coli K88 on day 6 (ETEC). Two groups (C− and LS−) remained unchallenged. On

day 18 and 19 of the study (12–13 days post E. coli challenge) the piglets were sacrificed and jejunal mucosa tissue scrapings were taken for isolation of RNA. Per group, equal amounts of RNA isolated from individual pigs (n = 12) were pooled. Messenger RNA expression levels in two RNA pools were compared by dual colour hybridisation. Micro array slides spotted with a library of expressed sequence tags (EST) prepared from jejunal mucosa were used (Niewold et al., 2005). Because RNA pools were prepared from relatively large number of piglets (n = 12) the possibility that mRNA's are detected that hybridize differentially due to inter-animal variation is limited. This paper focuses on differences in intestinal gene expression profiles observed between treatments LS− versus C− and treatments LSM+ versus LS+, which showed the most prominent differences in the first step analysis of the pooled samples. 3. Results and discussion Micro-array analysis showed that the abundance of pancreatitis associated protein (PAP) mRNA was eightfold higher in the pool prepared from LS− fed pigs than in the pool prepared from pigs fed with control (C−) diet (Table 1). PAP is a C-type lectin with anti-inflammatory properties and is expressed in intestinal epithelium by

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Paneth cells. The role of this protein in innate defence mechanism of the intestine is currently unknown. Recently, we showed that expression of PAP mRNA in jejunal mucosa increases rapidly (8 h after infection) upon exposure to ETEC and/or its toxins (Niewold et al., 2005) and Salmonella (Niewold et al., 2007). The results presented here suggest that transcription of this gene in the jejunum may also be enhanced by specific constituents of linseed. Interestingly, a recent study revealed that the level of expression of the PAP protein in the colon was significantly higher in mouse fed with a diet that contained 40% vegetables compared to a control diet without vegetables (Breikers et al., 2006). Similar as observed in the above mentioned ETEC and Salmonella experiments, expression of THO complex 4 mRNA (an oligomeric complex involved in transcription elongation by RNA polymerase II and crucial for transcription of certain coding regions) coelevated with PAP mRNA (Niewold et al., 2007). The abundance of hydrosteroid 17-β dehydrogenase 2 mRNA, a gene involved in the metabolism of androgen and estrogen hormones, was threefold lower in the LS− RNA pool than in the C− pool. Comparison of the pools prepared from ETEC challenged pigs (Table 1, LSM+ versus LS+) showed that the abundance of PAP mRNA in the pool prepared from pigs fed with defatted linseed (LSM+) was almost fourfold higher than in the pool prepared from pigs fed with full fat linseed (LS+). This could indicate that the fibre in linseed stimulates or maintains PAP transcription more effective than full fat linseed or that oil in full fat linseed reduces PAP transcription. In addition, to PAP the levels of mRNA's coding for GW112 protein (increased in inflamed colonic mucosa of humans with ulcerative colitis, function unknown), hCSM (a homologue to a transmembrane mucin, involved in the barrier function of epithelial tissues present on the apical membrane and intracellularly in goblet cells) and heparin binding protein 15/L22 (a 15 kDa-protein purified from mouse submandibular gland and bovine brain) were higher in the LSM+ pool than in the LS+ pool. Abundance of mRNA's coding for lactase-phorizin hydrolase (lactase), calbinding D-9k DNA (a vitamin D dependent calcium binding protein aiding in the transport of calcium across the cytoplasm of the enterocytes) and glutathione S-transferase (enzymes that utilize glutathione in reactions contributing to the transformation of a wide range of compounds including detoxification of substances) were lower in the LSM+ pool than in the LS+ pool. As pooled RNA preparations were compared on the microarray it should be noticed that the differences in gene

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expression profiles observed could be affected by variation in response between individual animals within a treatment group. Therefore, quantification of mRNA levels by quantitative real-time PCR in the jejunum of individual animals should give further insight into the meaning of the differences measured by this micro-array analysis. However, the observation that transcription of genes like hCSM and PAP, may be enhanced by feeding pigs with defatted or full fat linseed is most promising. PAP is specifically expressed in goblet and Paneth cells, cells responsible for the formation of mucus and other compounds of the innate defence of intestinal epithelium, respectively. Stimulation or inhibition of such genes by feeding piglets with a diet with specific functional constituents may be a convenient way to improve intestinal health under stressful conditions such as weaning. It can be concluded that diet composition does affect intestinal gene expression in post weaning piglets, either or not challenged with E. coli (ETEC). The intestinal micro-array technique is a promising tool to study the relationship between nutrition and feeding and gut tissue responses under defined conditions. The technique could likely in future be used in the development, application and evaluation of functional characteristics of feed ingredients, aiding the design of feeding concepts to support gut health. References Breikers, G., van Breda, S.G., Bouwman, F.G., van Herwijnen, M.H., Renes, J., Mariman, E.C., Kleinjans, J.C., van Delft, J.H., 2006. Potential protein markers for nutritional health effects on colorectal cancer in the mouse as revealed by proteomics analysis. Proteomics 6, 2844–2852. Fedeniuk, R.W., Biliaderis, C.G., 1994. Composition and physicochemical properties of linseed (Linum usitatissimum L.) mucilage. J. Agric. Food Chem. 42, 240–247. Niewold, T.A., Kerstens, H.H.D., Van der Meulen, J., Smits, M.A., Hulst, M.M., 2005. Development of a porcine small intestinal cDNA micro-array; characterization and functional analysis of the response to enterotoxigenic E. coli. Vet. Immunol. Immunopathol. 105, 317–329. Niewold, T.A., Veldhuizen, E.J.A., van der Meulen, J., Haagsman, H.P., de Wit, A.A.C., Smits, M.A., Tersteeg, M., Hulst, M.M., 2007. The early transcriptional response of pig small intestinal mucosa to invasion by Salmonella enterica serovar Typhimurium DT104. Mol. Immunol. 44 (6), 1316–1322. Oomah, B.D., 2001. Flaxseed as a functional food source. J. Sci. Food Agric. 81, 889–894. Pluske, J.R., Hampson, D.J., Williams, I.H., 1997. Factors influencing the structure and function of the small intestine in the weaned pig: a review. Livest. Prod. Sci. 51, 215–236. van Ommen, B., Stierum, R., 2002. Nutrigenomics: exploiting systems biology in the nutrition and health arena. Curr. Opin. Biotechnol. 13, 517–521.