key: cord-283257-rh3bxvv7 authors: Andrejčáková, Zuzana; Sopková, Drahomíra; Vlčková, Radoslava; Kulichová, Lucia; Gancarčíková, Soňa; Almášiová, Viera; Holovská, Katarína; Petrilla, Vladimír; Krešáková, Lenka title: Synbiotics suppress the release of lactate dehydrogenase, promote non‐specific immunity and integrity of jejunum mucosa in piglets date: 2015-12-21 journal: Anim Sci J DOI: 10.1111/asj.12558 sha: doc_id: 283257 cord_uid: rh3bxvv7 The aim of our experiment was to study how synbiotics are able to deal with the problems of post‐weaning piglets. Lactobacillus plantarum – Biocenol(TM) LP96 (CCM 7512), Lactobacillus fermentum – Biocenol(TM) LF99 (CCM 7514) and flaxseed (rich in n‐3 polyunsaturated fatty acids) were administered to 36 conventional piglets from a problematic breed with confirmed presence of enterotoxigenic Escherichia coli and Coronavirus. The experimental piglets were supplied with probiotic cheeses and crushed flax‐seed in the period starting 10 days before weaning and lasting up to 14 days post‐weaning. Piglets in the control group were supplied only control cheese. The impact of such additives on the release of lactate dehydrogenase (LDH; spectroscopic and electrophoretic assay), alteration of immunity (index of metabolic activity), jejunum histology (light microscopy), and health of conventional piglets from a problematic breed (monitoring of hematology, consistency and moisture of feces and body temperature) were examined. We found significant decrease in LDH leakage in the blood serum and tissue extracts, indicating better cell membrane integrity in the individual organs of animals. Probiotics and flaxseed applied together seem to be a good source of nutrients to improve the immune status and the integrity of jejunum mucosa during infection. © 2015 Japanese Society of Animal Science Diarrheic syndrome in weanlings is a serious health and economic problem in livestock production due to high morbidity and mortality. Nutrition and feeding of pigs, together with other environmental factors (breeding, reproduction, health status, rearing management) are among the economically most important factors influencing animal performance parameters and the overall economics of pig farming. Health and survival of neonatal piglets depend on the ability of intestinal mucosa to act as an effective barrier against various infectious agents. Enterotoxigenic Escherichia coli (ETEC) is the major reason for diarrhea in sucklings and weanlings, leading to excessive loss of fluids and electrolytes, resulting in profuse diarrhea, usually without histological lesions (Wilson & Francis 1986; Francis 2002) . Infections caused by Coronavirus (Porcine epidemic diarrhoea virus, PEDV; Mole 2013) are among the other diseases causing diarrhea and vomiting in this breed of piglets. Inflammation caused by ETEC may be effectively inhibited by the application of suitable immunomodulatory agents demonstrated in numerous experimental studies of piglets (Shirkey et al. 2006; Tohno et al. 2006; Walsh et al. 2008; Mizumachi et al. 2009; Chytilová et al. 2013) . Diet supplementation with probiotics has been shown to: (i) reduce the frequency of post-weaning diarrhea (Manner & Spieler 1997) ; (ii) play an important role in the intestinal barrier against inflammatory bowel disease (Laukoetter et al. 2008) ; and (iii) improve growth performance (Scheuermann 1993) in piglets. Furthermore, a strong immunomodulatory effect is also assigned to the n-6 and n-3 polyunsaturated fatty acids (PUFAs). The group of n-6 PUFAs have the ability to activate the immune system and act more as proinflammatories, whereas n-3 PUFAs have a significant anti-proliferative and anti-inflammatory effect (Yaqoob 2004) . The growth of pigs may be enhanced by the intake of extra n-3 PUFAs (Nguyen et al. 2003) , which have the capability to improve cell membrane fluidity (Stulnig et al. 2001; Russo 2009 ). The recent ban on the use of natural feeds has put pressure on the development of new feeding strategies and formulations to support performance and gut health. Many studies have demonstrated mostly microbiological and immunological improvements of ongoing infections caused by the additives mentioned above. The aim of our study therefore was to examine the effect of synbiotics through flaxseed feed supplementation in combination with Lactobacillus plantarum -Biocenol TM LP96 (CCM 7512) and Lactobacillus fermentum -Biocenol TM LF99 (CCM 7514), on the activity of lactate dehydrogenase (LDH), alteration of immunity, jejunum histology and health of conventional piglets from a problematic breed. The experiments with piglets (weanlings) were performed at the Institute of Microbiology and Gnotobiology, University of Veterinary Medicine and Pharmacy (UVMP) in Košice, Slovak Republic. The experiments were approved by the State Veterinary and Food Administration of the Slovak Republic (Approval No. 2519/10-221) and the animals were handled in a humane manner in accordance with the guidelines established by the relevant commission. The experimental animals were housed in stainless steel cages fitted with a slatted floor strewn with ¾ insulating rubber and ambient temperature of 20-22°C. The animals were divided into two groups: control C (n = 18, control cheese, sprinkle on the surface of feed) and LF group (n = 18, probiotic cheeses + crushed flaxseed). Throughout the study, the animals were fed diets mixed for early weaning of piglets OŠ-02 (Spišské Vlachy, Slovak Republic; Table 1 ) and had ad libitum access to water. The feed mixture was supplemented with crushed flaxseed (cultivar Flanders, AGRITEC, Czech Republic) as a source of PUFAs at a concentration of 10%. The fatty acid composition (percentage) of flaxseed was as follows: lipids (dry matter (DM) basis) -45.78; palmitic FA (C16:0) -5.1; stearic FA (C18:0) -3.7; oleic FA (C18:1) -18.4; linoleic FA (C18:2) -16.1; linolenic FA (C18:3) -56.8. In the period starting 10 days before weaning and lasting up to 14 days post-weaning, the experimental piglets in group LF were supplied with probiotic cheeses at a dose of 4 g/animal/day for each cheese, and in the same period the feed of group LF was supplemented with crushed flaxseed. Piglets in control group C were supplied control cheese at a dose of 8 g/animal/day. The Lactobacillus probiotic strains were isolated in the laboratory of the Institute of Microbiology and Gnotobiology, UVMP in Košice, Slovak Republic. The Lactobacillus plantarum -Biocenol TM LP96 (CCM 7512) strain originated from the gut contents of healthy suckling piglets. This strain was characterized by strong adherence to the epithelial cells from the porcine intestine, by inhibitory activity against E. coli O8:K88ab:H9 under in vitro conditions, and by production of hydrogen peroxide (Nemcová et al. 1997) . The Lactobacillus fermentum -Biocenol TM LF99 (CCM 7514) strain was isolated from the gastrointestinal tract of adult chickens. This strain was characterized by the growth in the presence of bile acids and gastric juice, sensitivity to antibiotics, inhibitory activity against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Düsseldorf, and aggregation and co-aggregation ability (Nemcová et al. 2003) . Cheddar cheese (chemical composition per 1 kg: proteins 23.8%, sugars 2.8%, lipids 30.1%, metabolisable energy 1.62 MJ) was used as a vehicle for the probiotic strains. The probiotic cheeses contained probiotic strains (each cheese contained one probiotic strain) at 1 × 10 9 cremoris) during typical Cheddar cheese production. The cheese that was used as a control was similar to cheddar cheese, but without the probiotic strains (referred to as control cheese). During the experimental period the piglets all underwent clinical observation. Health data were recorded twice daily (at 08.00 and 15.00 hours), specifically, body temperature (BT), consistency of feces (F) and moisture of feces (MF). Samples of feces were assessed visually using a scale from 1 to 5, where the number 1 meant solid feces, 2 paste, 3 sparse, 4 hydrous and 5 feces with an admixture of blood or mucus. The moisture of feces was determined by drying a sample of feces at 80°C to constant weight. Blood samples from piglets were taken from the plexus venosus suborbitalis on Day 0 (day of weaning; n = 6), Day 7 (n = 6) and Day 14 (n = 6) after weaning. Blood serum was separated from blood samples by centrifugation at 4°C and 1095 × g for 10 min for determination of total LDH (TLDH) and its isoenzymes (LDH 1 -LDH 5) expressed in μkat/L and for measurement of leukocytes. On sampling days, the piglets were humanely sacrificed (euthanased) using T61 a.u.v. (Intervet International BV, Boxmeer, Netherlands) intracardiac administration of 1 mL/kg/head. Heart, liver and skeletal muscle were collected from each pig and processed (tissue homogenates or extracts) until other analyses were done. The samples were cut into small pieces, and washed in buffered saline to remove excess blood and connective tissue. Two grams of tissue were then homogenized in 20 volumes of cold buffer (0.05 mol/L Tris-HCl buffer, pH 7.3) in line with Heinová et al. (1999) . Protein concentration was measured in accordance with Bradford (1976) and TLDH and its isoenzymes (LDH 1 -LDH 5) were expressed in international units per gram of protein (IU/g). Blood plasma was collected into tubes containing menadione ethylenediaminetetraacetic acid (K 3 EDTA) for hematological analysis using a BC-2008 VET automatic analyzer (Mindray, Shenzhen, China). The concentration of total LDH (μkat/L or IU/g) activity was assessed using commercial diagnostic kits (Randox, Crumlin, UK) with an Alizé automatic biochemical analyser (Lisabio, Pouilly-en-Auxois, France) at laboratory temperature. The measurement was conducted using light absorbance at 340 nm. For electrophoretic study, 10 μL of the blood serum and culture supernatant was used for each separation. A Hydrasys device (Sebia, Lisses, France) was used for the determination of LDH isoenzyme activities. The samples were separated using commercial Hydragel 7 ISO-LDH electrophoretic kits (Ecomed, Žilina, Slovak Republic) on alkaline-buffered (pH 8.4 ± 0.05) agarose gels (0.8g/dL). The dried gels were prepared for visual examination and densitometry to obtain accurate relative quantification of individual zones. The image of the electrophoretic migration was scanned by light transmission and automatically converted into an optical density curve presentation. Then photographs of the gels were taken. Qualitative evaluations of the gels were done directly from the electrophoretograms and the densitometric curves of the separations were created by means of Epson Perfection V 700 Photo densitometer scanning at 570 nm and evaluated using PHORESIS software (Version 5.50, 2009, Sebia, Lisses, France) . The iodonitrotetrazolium test (INT) -2-(4-iodophenyl)-5-fenyltetrazolium chloride -INT (Erba Lachema, Brno, Czech Republic)was carried out on microscale in accordance with the modifications made by Procházková et al. (1986) . The functional ability of phagocytes of metabolic processes occurring in phagocyting leukocytes (production of microbicidal substances, particularly H 2 O and O 2 ) based on the ratio between spontaneous activity and the activity after stimulation by zymosan (Sigma, St. Louis, MO, USA), that is, the index of metabolic activity (IMA), was assessed. Excisions from the jejunal mucosae (collected from piglets on Day 0 and Day 7) of 1 mm 3 size were fixed by immersion in 3% glutaraldehyde and postfixed in 1% osmium tetraoxide (both in 0.15 cacodylate buffer, pH 7.2-7.4). After dehydration in acetone, the excisions were transferred to propylene oxide, and embedded in Durcupan ACM (Fluka Chemie AG, Buchs, Switzerland). Semi-thin sections (250 nm) of specimens processed for transmission electron microscopy were cut on an LKB Nova ultramicrotome, stained with toluidine blue (Sigma-Aldrich, Bratislava, Slovak Republic) and examined under an Axio Lab. A1 light microscope (Zeiss, Jena, Germany) at 400× magnification. The data were assessed using one-way analysis of variance with Tukey's post hoc analysis (GraphPad Prism 3.0 for Windows; GraphPad Software, San Diego, CA, USA). Values listed in the tables and figures are the average values obtained from six samples. All data are means with standard error of mean (SEM). Differences within the group are marked with superscript letters ( a, b, c ) and considered to be significant at levels of a = P < 0.05, b = P < 0.01 and c = P < 0.001. Before the experiment was done, diagnostic analysis was conducted in the herd as kept by the animals' owner and samples of biological material were collected for laboratory analyses (bacteriological examination of rectal swabs, virological and parasitological examination of feces, hematological and biochemical examination of blood) which allowed us to diagnose: hypoproteinemia, lymphocytic leucocytosis and increased activity of bilirubin and enzymes (alanine aminotransferase, gammaglutamyltransferase). Infection with enterotoxigenic E. coli (Institute of Microbiology and Immunology, UVMP, Kosice, Slovak Republic) and Coronavirus (Vetservis, s.r.o., Nitra, Slovak Republic) was confirmed in fecal samples. During the experiment selected health parameters of the piglets were recorded as shown in Table 2 . The consistency of feces (F) in the control group of piglets deteriorated significantly (P < 0.001) during the experimental period, whereas in the experimental group the consistency had a more settled character. Moreover, the piglets with addition of synbiotics had better progress of infection in general. We noticed significant increase in the leukocyte count and hemoglobin (P < 0.01) after 14 days of the supplementation period. In the blood serum in the experimental group of piglets, significant decrease in TLDH concentration was noticed on Day 7 (P < 0.001) and Day 14 (P < 0.01) compared to Day 0 (Table 3 ). In the control group, without addition of probiotics and flaxseed, significant decrease was recorded on Day 14 (P < 0.001) compared to Day 7 after weaning. Average values of TLDH activity on Day 7 were significantly lower (P < 0.01) in the experimental group compared to control. Significant differences were also observed in the concentration of LDH 1 to 5 isoenzymes. All isoforms exhibited a decreasing trend at the end of the feeding period (Day 14) in comparison with the beginning of the supplementation period, and at different significance levels (see Table 3 ). Tissue extracts from the piglets' hearts (Table 4 ) showed significant differences between the observed groups in the concentrations of TLDH (P < 0.05) and LDH 1 (P < 0.01) on Day 14 after weaning. Overall, the excretion of LDH (TLDH and LDH 1-5) was markedly reduced on Day 14 in the experimental group compared to control. NS NS NS Note: C, control group; LF, group treated with probiotics and flaxseed; Day 0-14, study day after weaning; data are means ± SEM (IU/g); a mean values in rows with same superscript letters are statistically significant at the level of a = P < 0.05. The piglets' livers (Table 5 ) exhibited markedly decreased activity of TLDH and its isoenzymes on Day 14 after weaning at different levels of significance (P < 0.05; P < 0.01; P < 0.001). In the skeletal muscles of the piglets (Table 6) , we noticed significant decrease in TLDH, LDH 2, 3 and 5 (P < 0.05) on Day 14 after weaning when comparing the observed groups. The activity of LDH exhibited a decreasing trend at the end of the feeding period (Day 14). In our experiment, significant increase in non-specific immunity (IMA) was noticed on Day 7 (P < 0.01) and Day 14 (P < 0.05) after weaning when comparing the observed groups (Fig. 1 ). Significant increase (P < 0.05) was recorded in the index of metabolic activity in the experimental group of piglets. At the beginning of sample collection (Day 0 of the experiment) in the control group, the mucous layer of intestinal villi (Fig. 2) consisted of a single layer of columnar epithelial enterocytes (E) and goblet cells (GC). In the apical part of intestinal villi, small groups of dying enterocytes of irregular shape and with dark cytoplasm were present. Goblet cells did not show significant morphological changes. In the epithelium of intestinal villi numerous intraepithelial lymphocytes were present. Significant leukocyte infiltrations were observed mainly in the connective tissue of lamina propria mucosae. These findings contrasted with the histological sections of jejunum from the experimental piglets, where dying enterocytes occurred sporadically only in the apical part of intestinal villi. Microscopic changes in the mucous layer of the small intestine epithelium indicated persistent inflammation in the control group of weanlings (Day 7; Fig. 2 ). Intestinal villi were broad, and low and had strongly deformed shape. The presence of dying enterocytes was enormous, and the sparse connective tissue of lamina propria mucosae was significantly infiltrated by leukocytes, in contrast to the jejunum of the experimental group, where intestinal villi were high, of regular shape, and mucosa of the jejunum did not show any structural changes. Probiotics are live microorganisms which may contribute to the health of the host, when they are administered in appropriate amounts (FAO/WHO 2002) . Lactic acid bacteria regulate intestinal microbial homeostasis and the stabilizing function of the gastrointestinal barrier, while expressions of bacteriocins (Mazmanian et al. 2008 ) have immunomodulatory effect (Salzman et al. 2003) . They may inhibit procarcinogen enzymes and the ability of pathogens to colonize and infect the gut Note: C, control group; LF, group treated with probiotics and flaxseed; Day 0-14, study day after weaning; data are means ± SEM (IU/g); a,b,c mean values in rows with same superscript letters are statistically significant at the levels of a = P < 0.05; b = P < 0.01; c = P < 0.001. mucosa (Gill 2003) . Probiotic microorganisms act against infections of the gastrointestinal tract by means of their own production of bioactive molecules, such as organic acids, carbon dioxide, hydrogen peroxide, other low molecular weight substances and bacteriocins (Gomes et al. 2012) . Probiotics are able to displace pathogens such as Salmonella spp., Clostridium spp. and E. coli in the gastrointestinal tract of pigs through the mechanism of competitive inhibition for binding sites (Biernasiak et al. 2011) . The efficacy of probiotics may be potentiated by components of natural origin (so-called synbiotics) such as the essential PUFAs, which have impact on the nutrition of piglets (Tanghe & De Smet 2013; Tanghe et al. 2014) . The presence of PUFAs may positively improve the adaptation of piglets to the rapidly changing diet at weaning (Bomba et al. 2005; Marcinčák et al. 2009; Li et al. 2014) . They have direct regulatory action on leukocytes by binding to intracellular receptors or by modifying the release of second messengers (Klasing 1998) , and on red blood cell membrane fatty acid composition (Boehm et al. 1996) . It is known that LDH is a highly sensitive, but not specific marker of cell membrane disruption. Intracellular LDH (EC. 1.1.1.27), L-(+)-lactate: NAD + oxidoreductase leakage is a possible indicator of cell membrane integrity and cell viability (Legrand et al. 1992) . LDH catalyses the intraconversion of pyruvate and lactate and is involved in both the catabolism and anabolism of carbohydrates. In addition to metabolic roles in the cells, it includes a number of other biological processes (Powers et al. 1991) . The function of specific indicators is performed by LDH isoenzymes, which allow us to identify the damage to cells and tissues by different agents. LDH leakage from various cells (e.g. hepatocytes, monocytes, lymphocytes) into the environment is used as one of the markers of cytotoxicity for monitoring tissue and cell damage in in vitro conditions (Kopperschläger and Kirchberrger 1996; Šutiaková et al. 2004) . Moreover, mean values between columns with the same superscript letters are statistically significant at levels a = P < 0.05; b = P < 0.01. lactic acid level reflects the quantitative transformation of glycogen, and indicates typical or atypical processes of meat ripening (Koréneková et al. 2009 ). In our experiment, leakage of LDH into the interstitial space of organs was monitored in blood serum and tissue extracts from the heart, liver and skeletal muscle in piglets with persistent infection. In the group fed with synbiotics (combination of probiotics and PUFAs in the form of flaxseed) we noticed significant decrease in TLDH leakage and isoenzymes typical for individual tissues. Ghanem et al. (2005) experimentally infected mice with Schistosoma mansoni, and they found that after 14 days of probiotic administration (yogurt containing L. casei, L. plantarum, L. reuteri, L. acidophilus), the activity of LDH decreased. Similar results were recorded in our experiment with weanling pigs. The authors described the protective effect of probiotics through non-specific stimulation of the immune system. Otherwise, Rajput et al. (2013) described significant increase in concentration of LDH in the blood of pigs given feed supplemented with Bacillus subtilis. The concentration of TLDH and its isoenzymes in the liver of our piglets significantly decreased at the end of the feeding period, which indicated the bioprotective effect of synbiotics on hepatocytes. New studies (Imani Fooladi et al. 2013) have confirmed that probiotics have significant effect on the health status of the liver and the ability to treat various liver diseases. Based on our results, it could be assumed that the temporary increase in TLDH and its isoenzymes in the skeletal muscle of piglets, which otherwise continuously decreased, was probably due to the ongoing infection. Daugschies et al. (2000) noticed subsequent decrease in the activity of TLDH in muscles of calves after experimental infection with Sarcocystis cruzi, which may also be affected by enhancing the release of LDH into the bloodstream during ongoing infection. LDH is also produced by the parasites themselves, even though their presence in the blood of the host was not confirmed in this study. The impact of infection on serum LDH release was observed in the study by Nussinovitch et al. (2009) , who noticed an increase in TLDH and its isoenzymes LDH 4 and 5 in the blood of people with bacterial meningitis. It is well known that probiotics (Delcenserie et al. 2008; Trasino et al. 2013) and PUFAs (Harbige 2003) have the ability to improve the immune status of the organism. The index of metabolic activity (IMA) seems to be appropriate for testing the characteristics of nonspecific immunity in animals (Spišáková et al. 2009; Haladová et al. 2011) . In our study, the IMA index significantly increased in the group fed with lactobacilli and flaxseed, similar to the study of Wen et al. (2011) , who applied probiotics alone against rotavirus disease. The histological sections of jejunum from experimental piglets fed with synbiotics did not show any structural changes, in contrast to the control group of weanlings, where significant damage to intestinal villi was recorded, followed by lymphocyte proliferation. Many authors (Schroeder et al. 2006; Chandran et al. 2013; Núñez Currently, several studies aimed at improving the problems of pigs after weaning are based on adding healthpromoting substances or additives into animal feed. In our study, we point out the beneficial effect of probiotics (Lactobacillus plantarum and Lactobacillus fermentum) in combination with PUFAs (in the form of flaxseed) on animal health. Better integrity of cell membranes in the individual organs of animals was recorded by monitoring LDH release into the bloodstream and tissue extracts. Probiotics and flaxseed significantly improved the immune response to ongoing infection. In terms of morphology, we can conclude that the additives used had a positive effect on the course of infection and renewal of the enterocyte cell membrane. 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We would like to thank Ing. M. Kozackova for the technical assistance at electrophoresis and Mr. A. Billingham for English correction of the manuscript.