Pa1820 - Hogeschool Gent

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J. Michiels et al. Intact brown seaweed for pigletsTable 1 Ingredient and analysed and calculated nutrient compositionof the pre-starter (days 0–4) and the experimental starter diets (days5–28) for weaned pigletsPre-starterStarter*CON SW2.5 SW5.0 SW10.0Ingredient composition (g/kg as fed)Oats heat-treated 100Corn heat-treated 220 156 156 156 156Barley 343 343 343 343Wheat 50.0 157 155 152 147Soya meal 44/7 98.0 98.0 98.0 98.0Potato protein 4.9 4.9 4.9 4.9Full-fat soya beans 98.0 98.0 98.0 98.0Soybean flour 200 lm 140 14.7 14.7 14.7 14.7Whey powder 90.0 36.8 36.8 36.8 36.8Lactose powder 60.0 14.7 14.7 14.7 14.7Soybean oil 40.0 14.7 14.7 14.7 14.7Sodium chloride 4.6 4.6 4.6 4.6Monocalcium-phosphate 5.4 5.4 5.4 5.4Limestone 5.9 5.9 5.9 5.9Closed formula300premix pre-starterClosed formula36.8 36.8 36.8 36.8premix starteràCelite 545 coarse 10.0 10.0 10.0 10.0Dried marine seaweed2.5 5.0 10.0(A. nodosum)Analysed nutrient comp. (g/kg as fed)DM 924 888 888 887 888CAsh 51 56 57 58 58CP 189 181 178 178 176EE 86 57 56 57 57CF 17 30 30 31 32Starch 240 383 387 380 379Sugar 217 61 60 61 59NDF

Intact brown seaweed for pigletsJ. Michiels et al.saline and fixed in neutral buffered formalin for24 h pending further measurements of histo-morphologicalparameters.Analyses of gut bacteria and metabolitesBacteria in digesta and excreta were determinedusing selective media (stomach, S1, S2 and excreta)and molecular techniques (S2 and caecum). Thering-plate technique (Van Der Heyde and Henderickx,1963) was used to count the bacteria (viablecounts; log 10 CFU/g fresh digesta) in digesta ofstomach, S1 and S2. Ten-fold dilutions were madefrom 1 g aliquots of fresh digesta, using a sterilizedpeptone solution (1 g peptone + 0.4 g agar + 8.5 gNaCl in 1 l aq. dest.) and plated onto selectivemedia in duplicate. Selective media were used forcounting the following bacterial groups: total anaerobicbacteria (Reinforced Clostridial Agar, CM0151,Oxoid, Basingstoke, UK + 0.001% hemin; incubatedfor 48 h at 37 °C anaerobically), coliform bacteria(Eosin Methylene Blue Agar, CM0069, Oxoid; incubatedfor 24 h at 37 °C aerobically), Escherichia coli(Tryptone Bile X-glucuronide Medium, CM0945,Oxoid; incubated for 24 h at 37 °C aerobically);streptococci (Slanetz & Bartley Medium, CM0377,Oxoid; incubated for 48 h at 37 °C aerobically), lactobacilli(Rogosa Agar, CM0627, Oxoid + 0.132%acetic acid; incubated for 48 h at 37 °C anaerobically)and Bifidobacteria (Tryptone Peptone Yeastextract, Scharlau Chemie S.A., Barcelona, Spain;incubated for 72 h at 37 °C anaerobically; only S2digesta).Molecular analysis (PCR-DGGE) was applied tosamples from S2 and caecum. Initially DNA wasextracted from the cells by use of the QIAGENDNeasy Ò DNA stool mini kits (Qiagen, Hilden, Germany).The manufacturer’s methodology for DNAisolation from stools for pathogen detection wasadhered, i.e. samples were incubated at 95 °C for5 min to lyse all bacteria, including the more temperature-resistantGram-positive bacteria (manufacturer’sguidelines), and to maximise the DNAretrieval yield. The DNA concentration and A 260 /A 280 ratio was measured using a NanoDrop 1000(Thermo Scientific, Rockfor, IL, USA). Fragments of16S rRNA genes were amplified from the extractedDNA by PCR using universal bacterial primers338CG: 5¢-CGC CCG CCG CGC GCG GCG GGC GGGGCG GGG GCA CGG GGG GCC TAC GGG AGGCAG CAG-3¢ and RP534: 5¢-ATT ACC GCG GCT GCTGG-3¢ with the GC-clamp present to stabilise themelting behaviour of DNA fragments (Muyzer et al.,1993). Primers were synthesized by Generi BiotechLtd (Hradec Králové, Czech Republic). PCR conditionsused were as follows: denaturation (3 min94 °C), 35 cycles (1 min 94 °C, 30 s 55 °C, 1 min72 °C), and final elongation (10 min 72 °C) (Muyzeret al., 1993). PCR reaction (30 lL) was performedusing RED Taq ReadyMix with MgCl 2 (Sigma-Aldrich, Munich, Germany) and contained 30 ng ofthe DNA template and 1 lM of each primer. Theresulting amplicons were analysed on a 2% (w/v)TAE agarose gel to check for PCR products of thesize predicted (233 bp; Muyzer et al., 1993). Ampliconresolution was performed using denaturing gradientgel electrophoresis (DGGE) using the BioRadDcode Universal Mutation Detection System (Bio-Rad, Hercules, CA, USA), following the manufacturer’sguidelines. PCR products (25 ll) were loadedonto 9% TAE polyacrylamide gels, which containeda 35–60% denaturant gradient (100% denaturant:7 M urea and 40% deionised formamide). Electrophoresiswas performed in 1· TAE (40 mmol/l Tris,20 mmol/l acetic acid, 1 mmol/l EDTA) buffer at aconstant voltage and temperature of 55 V and 60 °Cfor 19 h. Gels were then stained for 30 min withSYBR Green I dye, 10 ppm (Mrazek et al., 2008)and the gel image saved with a EC3 gel documentationsystem (UVP Bioimaging Systems, Upland, CA,USA). Prior to the DGGE of all samples, the standardladder was created by individually PCR-amplifiedDNA of Escherichia coli ATCC 25922, Salmonella entericaserovar Enteritidis CCM 4420, Bifidobacteriumbifidum ATCC 29521, Enterococcus faecalis ATCC11700, Streptococcus salivarius ATCC 7073, Lactobacilusacidophilus ATCC 4356, Lactobacillus johnsonii ATCC33200, Bacteroides suis ATCC 35419 using 338CG andRP534 primers. After confirmation of individual PCRproducts formation for each strain, productswere combined in equal ratios and diluted 1:1 withDGGE loading buffer (Simpson et al., 2000). DGGEprofiles were compared using similarity trees. Briefly,each band position present in the gel was binarycoded for its presence or absence within a lane(McEwan et al., 2005). The degree of similarity wascalculated using the Nei and Li/dice similarity index(Nei and Li, 1979; Baránek et al., 2010) anddendrogram was constructed by the Neighborjoiningmethod with bootstrap analysis by 1000repeats using the FreeTree program (Pavlicek et al.,1999). Similarity trees were further adjusted usingthe FigTree program (Tree Figure Drawing Tool,version 1.3.1, 2006–2009, Andrew Rambaut,Institute of Evolutionary Biology, University ofEdinburgh).4 Journal of Animal Physiology and Animal Nutrition ª 2011 Blackwell Verlag GmbH

J. Michiels et al. Intact brown seaweed for pigletsBacterial metabolites were determined in digestaof S2 and caecum. Short chain fatty acids (SCFA)and lactic acid were analysed by a GC methoddescribed by Jensen et al. (1995) and modified byMissotten et al. (2009).Morphological characteristics of small intestineMeasurement of the morphological parameters villuslength (V) and crypt depth (C) and for samples of J1and J2 were carried as described by Van Nevel et al.(2003). Briefly, after dehydration and impregnationwith paraffin, 4 lm sections (six per piglet and intestinalsite) were stained with haematoxylin. V (fromtip to base) and C (from base to opening) of all wellorientedvilli and adjacent crypts were measuredusing a microscope equipped with a camera andcomputer with appropriate software (Olympus,CX41/U30, software CellB, Aartselaar, Belgium). Atleast 20 couples V–C were measured per piglet andintestinal site.from voltage deflections (PD; mV) in response tobipolar 50 lA current pulses generated for 200 ms.The measured PD and corresponding Rt, calculatedby the Ohm’s law were given every 6 s. After anequilibration period of 10 min, tissues were shortcircuited.The short-circuit current (Isc; lA/cm 2 ) wasassumed to be an indirect measure of net transcellularelectrolyte movement. Basal values (Rt BASAL ,X cm 2 ; Isc BASAL , lA/cm 2 ) were obtained as averagevalues between 15 and 20 min. At 20 min,16 mmol/l D-glucose was added to the mucosal sideand simultaneously 16 mmol/l mannitol was addedto the serosal side in all chambers. Twenty minuteslater, 5-HT (0.1 mmol/l) was added to the serosalside. Finally, 20 min after adding 5-HT, 5 mmol/ltheophylline was added bilaterally. The change in Isc(DIsc; lA/cm 2 ) due to addition of D-glucose and secregatogueswas calculated by subtracting the Iscbefore stimulation from the peak response after stimulationwhich usually occurred within 10 min afterthe addition.Absorptive and barrier function of small intestineThe intestinal segments were rinsed with a Ringer’sbuffer solution and then placed in an oxygenatedRinger’s buffer solution at 38 °C. The Ringer’s solution(pH 7.4) contained (in mmol/l): 115 NaCl, 25NaHCO 3 , 0.4 NaH 2 PO 4 ÆH 2 O, 2.4 Na 2 HPO 4 Æ2H 2 O, 5KCl, 1.2 CaCl 2 Æ2H 2 O, MgCl 2 Æ6H 2 O and 6 D-glucose.The segments were opened longitudinally along themesenteric border. The epithelium was stripped of itsserosal and muscle layers and then two sheets weremounted in two modified Ussing chambers (acrylatepolymer) (Dipl.-Ing. Mubler Scientific Instruments,Aachen, Germany). Edge damage was minimised byplacing silicon sheets on both sides of the tissue. TheUssing chambers had an opening area of 1.07 cm )2 .All tissues were mounted within 15 min followingeuthanisation. The following buffer solution (pH 7.4)was added to the serosal side of the epithelium (inmmol/l): 115 NaCl, 25 NaHCO 3 , 0.4 NaH 2 PO 4 ÆH 2 O,2.4 Na 2 HPO 4 Æ2H 2 O, 5 KCl, 1.2 CaCl 2 Æ2H 2 O, MgCl 2 Æ6H 2 O and 12 D-glucose. On the mucosal side,12 mmol/l D-glucose was replaced by 12 mmol/lmannitol. The temperature of buffer solutions washeld constant at 38 °C and mixed and gassed with a95% O 2 and 5% CO 2 mixture by a gas lift system.Electrical measurements were obtained by a microcomputer-controlledvoltage/current clamp (Michielset al., 2010). The electrophysiological measurementswere started under open circuit conditions. Thetransmucosal resistance (Rt; X cm 2 ) was determinedParameters of oxidative statusThe FRAP assay is considered as a measure of the‘total antioxidant power’, referred analogously to asthe ‘ferric reducing ability of plasma’, according tothe method described by Benzie and Strain (1996).The test is based on the reduction of the ferrictripyridyltriazine(Fe III -TPTZ) complex to the ferrous(Fe II ) form at low pH. The malondialdehyde (MDA)concentration in plasma was measured by theTBARS method (thiobarbituric acid reactive substances),as described by Grotto et al. (2007), to asseslipid oxidation. Glutathione Peroxidase (GSH-Px, EC1.11.1.9) activity was determined in plasma accordingto the method described by Hernández et al.(2004). The a-tocopherol concentration in plasmawas determined by HPLC using a-tocopherol as standardsolution, as described by Desai (1984). Resultsare expressed as lg ofa-tocopherol/ml plasma.Chemical analysesDiet and freeze-dried pooled (per treatment) contentof digesta of S2 and rectum were used to quantify4 mol/l HCl insoluble ash as indigestible marker tocalculate apparent digestibility coefficients. Thismarker was also used to calculate the apparentdigestibility (%) of nutrients the formula: 100 -(N comp · M feed )/(N feed · M comp ) · 100, in whichN comp and M comp are the concentration of respectivelythe nutrient and the marker in the respectiveJournal of Animal Physiology and Animal Nutrition ª 2011 Blackwell Verlag GmbH 5

Intact brown seaweed for pigletsJ. Michiels et al.section of the GIT and N feed and M feed are the concentrationof respectively the compound and themarker in the feed. Analysis of dry matter, crudeash, crude protein, ether extract and crude fibrewere determined according to EU standard methodsas indicated by Van Nevel et al. (2003).Statistical analysisData from the plate countings were log 10 transformed.All data were analysed for normal distributionby the one-sample Kolmogorov–Smirnov test.In all cases data were normally distributed. Then,the effect of treatment was tested using linear modelsin which group of piglets was used as blockingfactor. In none of the case there was an interactionbetween treatment and group of piglets, hence interactionwas not included in the models. Animal wasused as the experimental unit for analysis of bodyweight (BW, kg) and average daily gain (ADG,g/day; using initial body weight as covariate)(n = 32–40 per treatment). Pen was used as theexperimental unit for analysis of average daily feedintake (ADFI; g/day), feed conversion (F:G) andparameters from the killed animals; the latter referringto one animal per pen (n = 8 per treatment).For analysis of faeces score data the repeated measureswere included as within-subject factor. Treatmentsmeans were compared by the post hoc Tukeytest. All calculations were carried out using the SPSS15.0 program for Windows (SPSS, Chicago, IL, USA).ResultsThroughout the experiment the health status of theanimals was satisfactory and animals did not sufferfrom severe diarrhoea. Piglets fed 10.0 g/kg SWshowed more firm excreta compared to control animals(p < 0.05) (Table 2). However, treatment hadno effect on any of the performance parameters monitored(Table 2). Bacteriological analysis of pooledexcreta sampled on days 26 and 27 did not show differencesbetween treatments (data not shown).Apparent ileal and faecal nutrient digestibility wascomparable among treatments (data not shown). SWsupplementation did not affect the digesta characteristics(pH, fresh matter weight and dry matter content),except for pH of S1 digesta there was atreatment effect (p < 0.05). Neither did SW supplementationalter the number of bacteria in the differentsections of the GIT (Table 3). Dendogramsobtained when DGGE analysis was performed inDNA extracted from the piglets did not show anyTable 2 Effect of dried marine seaweed (A. nodosum) supplementationon BW, ADG, ADFI, F:G and faeces score of weaned piglets in aperformance trial (eight pen replicates, four to five animals per pen)*Itemgrouping of objects within a particular treatment,neither in S2 and caecal digesta (data not shown).Bootstrap values were low, suggesting the low uniformityof samples within treatments, i.e. high diversityamong individuals. The addition of the seaweedinto the diet did not show any effect on the compositionof bacteria in S2 and caecum of piglets. Treatmenthad no biological relevant or statisticallysignificant effect on histo-morphological and electrophysiologicalparameters (data not shown) which isin accordance to the lack of effect considering animalperformances and gut bacteria. Oxidative status measuredby the TBARS and FRAP method, the GSH-Pxactivity and a-tocopherol concentration was notchanged by feeding SW (Table 4).DiscussionExperimental dietCON SW2.5 SW5.0 SW10.0SEMàp-ValueDays 5–11Final BW (kg) 7.14 7.06 7.04 7.04 0.069 0.666ADG (g/day) 60 53 49 45 7.7 0.558ADFI (g/day) 166 157 158 161 12.5 0.958F:G 3.47 3.74 2.52 2.10 0.670 0.354Days 12–28Final BW (kg) 11.91 11.62 11.37 11.74 0.268 0.539ADG (g/day) 286 274 259 282 13.6 0.518ADFI (g/day) 471 445 430 457 18.8 0.475F:G 1.65 1.63 1.68 1.62 0.042 0.781Days 5–28Initial BW (kg) 6.72 6.69 6.70 6.72 0.034 0.842Final BW (kg) 11.91 11.62 11.37 11.74 0.268 0.539ADG (g/day) 220 209 198 213 10.6 0.542ADFI (g/day) 381 360 350 369 14.6 0.488F:G 1.74 1.73 1.78 1.74 0.040 0.871Faeces score§ 4.1 a 4.4 ab 4.3 ab 4.8 b 0.16 0.019*Piglets were fed a pre-starter diet from days 0 to 4 and a starter dietfrom days 5 to 28; four different starter diets were tested: a controldiet (CON), CON + 2.5 g dried seaweed per kg (SW2.5), CON + 5.0 gdried seaweed per kg (SW5.0) and CON + 10.0 g dried seaweed perkg (SW10.0).Values with different superscripts within a row are significantly differentat p < 0.05 (a,b).àSE of least squares mean.§Faeces were visually checked every 2 days starting from day 5 andcoded per pen on a scale ranging from 0 (liquid and extremely softexcreta) to 6 (hard faeces).The aim of the present study was to assess the effectsof intact dried A. nodosum on animal performances,6 Journal of Animal Physiology and Animal Nutrition ª 2011 Blackwell Verlag GmbH

Intact brown seaweed for pigletsJ. Michiels et al.in the SW10.0 diet of the current study is equal orhigher than that of Gahan et al. (2009) respectively(calculation based on data of Moen et al., 1997;MacArtain et al., 2007); which leads to the presumptionthat other components than laminarin andfucoidan might counteract the effects of laminarinand fucoidan. This has also been suggested by Gardineret al. (2008) who found a linear decrease inADG of grower–finisher pigs as the level of anA. nodosum extract (3–9 g/kg) increased. Theseauthors suggested three possible reasons for that: (i)the presence of phlorotannins decreasing feed intakeand N-retention, (ii) excess of potassium intake causingan anion–cation imbalance, which might largelydepend on the type of extract and (iii) high levels ofalginates that might increase digesta viscosity. Wedid not observe any effect on feed intake and apparentN-digestibility. However, tannins may also interactwith minerals and decrease their bioavailability.The dry matter content of the digesta, which is anindicator for digesta viscosity was not differentamong treatments. Finally, the seaweed supplementationrate (2.5–10 g/kg) and timing (8/9 days) mightbe too low and too short respectively to provoke aprebiotic effect. Indeed, higher inclusion rates of severaltypes of pure non-digestible oligosaccharides andnon-starch polysaccharides were needed to deliver a(substantial) shift in the microbial ecology in the GIT(e.g. fructooligosaccharides and transgalactooligosaccharides,40 g/kg, Houdijk et al., 2002; inulin, 50–150 g/kg, Wellock et al., 2007; alginate, 1 g/kg andinulin, 15 g/kg, Janczyk et al., 2010). In addition, inpiglets two weeks post-weaning the hindgut fermentativecapacity is rather limited and as argued abovethe general fermentability of seaweed carbohydratesis variable and limited. The reducing effect on E. coligrowth in the foregut observed in previous work (Diericket al., 2009) could not be confirmed here. Thismight be due to differences in experimental designor basal diet composition. In opposite to the study ofDierick et al. (2009), here dairy products (wheypowder and lactose, 50 g/kg feed) were included inthe basal diet. Lactose is a major substrate for particularmicrobial groups (e.g. lactobacilli) and hencethe bacterial gut composition might have beenaltered. This is evidenced by the higher numbers ofstreptococci and lactobacilli in gastric and S1 digestain the current study compared to those found by Diericket al. (2009), while the number of total anaerobicbacteria were equal in the control treatments ofboth studies. This may result in depressive effects oncoliform growth through the production of bacteriocinsor a reduction in digesta pH. This leaves lessroom for steering/improvement of gut microbiotacomposition. In line with this, Gahan et al. (2009)showed that the growth-enhancing effect of an A.nodosum extract diminished when the lactose contentin the diet was increased. However, the lactose levels(60–250 g/kg) in that experiment were (much)higher than in the current experiment (approximately40 g/kg). Gardiner et al. (2009) also reporteddecreased ileal coliform counts in finishing pigs. Inthe current study the profile of bacterial metabolitesin S2 or in the caecum, neither the bacterial compositioninvestigated by PCR-DGGE analysis of bacterialDNA was changed. Nevertheless, the SW10.0showed significantly more firm excreta which mightbe related to differences in fibre fermentation orwater retention in the colon. So far, effects on thegut bacteria are not conclusive and perhaps largelydepending on basal diet composition, type of seaweed,extraction procedure, chemical nature of components(e.g. Rioux et al., 2007) and age and healthstatus of animals.In addition to the pronounced temporal andlong-lasting adaptations of the gut histo-morphologyand function, piglets suffer from oxidative stressafter withdrawal from the mothers’ milk and separation.A. nodosum contains several classes of antioxidants(phlorotannins, tocopherols, ascorbic acidand carotenoids) and therefore provide a dietarymeans that might prevent or diminish oxidativestress after weaning. As outlined above, A. nodosumsupplementation did not affect a range of markersof plasma oxidative status. Here, no particular challengewas imposed to induce oxidative stress (e.g.high content of polyunsaturated fatty acids or heatstress). Therefore it can be assumed that commondietary levels of synthetic antioxidants (i.e. ethoxyquinand BHT, 38 and 75 mg/kg respectively) preventedlipid peroxidation in feed and digestaadequately. Also, the vitamin A and E contents indried A. nodosum (Moen et al., 1997; MacArtainet al., 2007) in the SW diets did not add much tothe dietary level of these vitamins from the controldiet (15 000 IE and 100 mg/kg for vitamin A and Erespectively). The control diet did not contain supplementalvitamin C but the addition of vitamin Cto weaner diets is yet controversial (e.g. Fernandez-Duenas et al., 2008). In this respect, higher doses ofdried A. nodosum than the once we investigated(the highest SW level in this study was 10.0 g/kg)could be appropriate. However, dried A. nodosumcontains high amounts of iodine (Dierick et al.,2009) and hence by including higher SW levels themaximum EU-allowed iodine content in complete8 Journal of Animal Physiology and Animal Nutrition ª 2011 Blackwell Verlag GmbH

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