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Possible molecular mechanisms by which an essential oil blend from star anise, rosemary, thyme, and oregano and saponins increase the performance and ileal protein digestibility of growing broilers Henry Reyer, Jürgen Zentek, Klaus Männer, Ibrahim M.I. Youssef, Tobias Aumüller, Julian Weghuber, Klaus Wimmers, and Andreas Müller J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01925 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 22, 2017

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Journal of Agricultural and Food Chemistry

Possible molecular mechanisms by which an essential oil blend from star anise, rosemary, thyme, and oregano and saponins increase the performance and ileal protein digestibility of growing broilers Henry Reyer†,&, Jürgen Zentek‡,&, Klaus Männer‡, Ibrahim M.I. Youssef‡,§, Tobias Aumillerǁ, Julian Weghuber⊥, Klaus Wimmers†,#,*, Andreas S. Muellerǁ †

Leibniz Institute for Farm Animal Biology (FBN), Institute for Genome Biology, WilhelmStahl-Allee 2, 18196 Dummerstorf, Germany

‡

Free University of Berlin, Department of Veterinary Medicine, Institute of Animal Nutrition, 14195 Berlin, Germany

§

Beni-Suef University, Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Beni-Suef 62511, Egypt

ǁ

Delacon Biotechnik, 4221 Steyregg, Austria

⊥

University of Applied Sciences Upper Austria, Institute for Food Technology and Nutritional Sciences, 4600 Wels, Austria

#

Faculty of Agricultural and Environmental Sciences, University Rostock, 18059 Rostock, Germany

&

These authors contributed equally to this work as first authors.

*

Corresponding author Prof. Dr. Klaus Wimmers Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany Email: [email protected] Tel.:

+49 (0) 38208-68-600

Fax:

+49 (0) 38208-68-602

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ABSTRACT

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Phytogenic feed additives represent a potential alternative to antibiotics with attributed health

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and growth promoting effects. Chickens supplemented with an essential oil blend, a Quillaja

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saponin blend or a combination of both phytogenic preparations showed a comprehensively

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and significantly improved apparent ileal digestibility of crude protein and amino acids

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compared to control birds. Accordingly, holistic transcriptomic analyses of jejunum and liver

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samples indicated alterations of macromolecule transporters and processing pathways likely

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culminating in an increased uptake and metabolizing of carbohydrates and fatty acids.

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Complementary analyses in Caco-2 showed a significant increase in transporter recruitment to

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the membrane (SGLT1 and PEPT1) after addition of essential oils and saponins. Although the

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penetrance of effects differed for the used phytogenic feed additives, the results indicate for

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an overlapping mode of action including local effects at the intestinal border and systemic

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alterations of macronutrient metabolism resulting in an improved performance of broilers.

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Key words: Digestibility, saponin, essential oil, SGLT1, PEPT1, gene expression


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INTRODUCTION

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In the EU in 2006 the ban on the low dose admixture of antibiotics as growth promoting

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agents into the feed of farm animals has expedited research activities for suitable natural

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alternatives with similar beneficial effects. Among these alternatives, phytogenic feed

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additives represent a promising but heterogeneous class of supplements which may promote

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health and performance of farm animals, including poultry.1,2 Palatability of feed and higher

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ileal nutrient digestibility, possibly through increased pancreatic enzyme secretion, are

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considered as mechanisms for growth promoting effects of phytogenic feed additives.3,4

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Moreover, beneficial effects of these additives on performance were referred to their

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favorable influence on the intestinal microbiota5, on antioxidative protection systems,6,7 on the

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immune system, and on the anti-inflammatory responses of animals.8

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Previous studies investigating the supplementation of broiler diets with phytogenic feed

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additives produced inconsistent results.1 Whereas some researchers reported a significant

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increase in performance and ileal nutrient digestibility due to feeding herbs or essential oil

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blends,9,10 others could not demonstrate an impact on feed intake, weight gain, feed

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conversion or nutrient digestibility.11-13 The contradictory results of these studies may derive

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from different factors including the parts of the plants used for the additives, the physico-

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chemical properties, the content of active substances, the method of processing, and the

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interaction with other feed components.4,14

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As mentioned above, the current literature provides several lines of evidence for beneficial

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effects of phytogenic feed additives on immune system and digestive functions. Up to date,

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the specific mode of action is still scientifically interesting but, despite of a long history of

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use, remains unclear for most phytogenic products. The objective of the present study was to

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investigate the effects of a defined and standardized mixture of essential oils, Quillaja

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saponins, and of a combination of both phytogenic preparations on the performance and on 3 ACS Paragon Plus Environment


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ileal nutrient digestibility of growing broilers. This essential oil- and saponin combination is

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part of Delacons phytogenic feed additive Biostrong 510 EC which was most recently

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approved in the European Union as zootechnical feed additive, according to regulation

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1831/2003. Moreover it is subject of the European Patent 15183332.4. In order to investigate

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possible molecular mechanisms that mediate the effects on performance and nutrient

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digestibility, differential gene expression patterns in jejunum and liver were studied using

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microarray technology. Moreover, in-vitro models employing a human colon cancer cell line

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(Caco-2) were used to investigate the involvement of nutrient transporters. Specifically, the

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effects of the essential oil mixture, saponins, and the combination of both preparations on the

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levels of sodium glucose transporter 1 (SGLT1) and peptide transporter 1 (PEPT1) were

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studied.

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MATERIALS AND METHODS

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Reagents and Materials

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ALEXA-fluorochrome-coupled antibodies for SGLT1 (bs-1128R-A647) and PEPT1 (bs-

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10125R-A647) were purchased from Bioss Antibodies (Woburn, MA, USA). Cell culture

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media and supplements including Minimum Essential Medium (MEM) with Earle’s salts,

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Dulbecco's

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penicillin/streptomycin (P/S) were purchased from Invitrogen (Lofer, Austria). The enterocyte

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differentiation medium (EDM) and MITO+ Serum Extender (MITO) were obtained from BD

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Bioscience (BD Bioscience, Bedford, MA). All other chemicals were of molecular biology

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grade, unless noted otherwise.

Modified

Eagle

Medium

(DMEM),

fetal

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Housing of Birds and Diets


bovine

serum

(FBS)

and

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One hundred and forty-four one-day-old healthy male broiler chickens (Cobb 500) were

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obtained from a commercial hatchery (Cobb Germany, Avimex GmbH, Spreenhagen,

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Germany). The birds were randomly assigned to the four experimental groups, consisting of

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36 chicks each. The experiment lasted for 21 days. Birds were housed in balance crates with 4

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birds per crate and 9 pens per treatment. The pen represented the experimental unit. The

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poultry house was provided with controlled climate and forced ventilation (0.3 to 0.6

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m3/hour/kg body weight). Room temperature was gradually reduced from 32°C at day 1 to

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28°C at day 21 of age. The relative humidity was maintained between 50 and 65 %. The

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lighting period with artificial light (20 lux) lasted for 24 h during the first four days and was

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reduced to 18 h from day 5 onwards. Feed was offered ad libitum via automatic feeders. Fresh

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water was continuously supplied by nipple drinkers. Health status of broilers was checked

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daily by a veterinarian. All animal procedures followed the national guidelines and were

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approved by the Free University of Berlin Animal Care Committee.

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The birds were fed on a starter diet during the 21 days-experimental period. The diets were

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calculated to meet the nutrient requirements for broiler chickens, recommended by the Society

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of Nutrition Physiology.15 The diets were fed in mash form and included no interfering

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additives such as enzymes, growth promoters, antibiotics or coccidiostats.

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The experimental diets contained either no phytogenic feed additives (Control) or were

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supplemented with 25.0 mg/kg of an essential oil blend from star anise, rosemary, thyme, and

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oregano (Essential oils), 46.0 mg/kg of a Quillaja saponin blend (Saponins), or a combination

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of both phytogenic preparations (Essential oils + Saponins). The phytogenic preparations

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consisted of the plant extracts, 0.1 kg silica and 0.2 kg limestone made up to 1 kg with fine

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wheat bran. They were supplemented in form of premixes into the diets. Furthermore, 5 g/kg

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titanium dioxide (TiO2) was added to the diets as an indigestible marker to determine the

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apparent ileal digestibility. Formulated ingredients and calculated nutritional composition of

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the basal diet is given in Table 1. All experimental diets were analyzed for the nutritional 5 ACS Paragon Plus Environment


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composition using standard methods (Table 2).16 Analyzed nutrient concentrations of the

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experimental diets were in line with the calculated nutrient concentrations. Furthermore,

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experimental diets were analyzed for their concentration of thymol which is considered as the

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leading substance in the essential oil blend (Table 3). The expected thymol concentration was

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1.0 mg/kg complete diet. Since Quillaja saponins contain a broad spectrum of up to 15

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different saponins, the relative saponin concentration in the premixes of the Saponins and

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Essential oils + Saponins groups was determined by their foaming properties.17 To ensure a

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homogeneous distribution of premixes in the complete diets, a magnetic microtracer was

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added to the premixes and its recovery was determined in diets. The analyses of the foam

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index in the diets of Saponins and Essential oils + Saponins groups, as well as of the magnetic

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marker in the complete diets of these groups were in accordance with the expected values. As

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expected, no thymol was detected in the complete diets of Control and Saponins groups.

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Performance of Broilers

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The growth performance of birds (body weight, body weight gain, feed intake, and feed

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conversion ratio) was determined at the start and at the end of the experiment as well as at

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weekly intervals per pen, representing the experimental unit. For calculation of the individual

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body weight gain, feed intake, and feed conversion, the data was corrected for the number of

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dead or culled chicks.

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Tissue Sampling and Measurement of Apparent Ileal Nutrient Digestibility

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At the end of the experiment (d 21 of age) and about five hours after starting the lighting

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cycle, the birds were stunned and subsequently sacrificed by decapitation. For 10 broilers per

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treatment (2 batches of 5 birds each), liver samples (0.5 g from the cusp of the right liver

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lobe) and a 20 cm part of the jejunum, located approximately 20 cm after the end of duodenal

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loop, were dissected. The jejunum section was opened longitudinally, flushed with sterile 6 ACS Paragon Plus Environment

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saline, and the mucosa was scraped off. All samples were snap frozen in liquid nitrogen, and

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stored at -80°C until RNA extraction. Moreover, the apparent ileal digestibility of nutrients

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was analyzed for 27 broilers per treatment (3 broilers per balance crate). Therefore, the ileal

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digesta was collected by purging an intestinal segment, from Meckel’s diverticulum to the

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ileo-ceacal junction, with 5 mL of aqua bidest. The ileal digesta of 3 birds of a balance crate

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were pooled in 50 mL tubes and immediately stored at -20°C until analysis. The digesta were

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freeze-dried, ground to pass through a 0.25-mm mesh, and mixed thoroughly before chemical

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analysis. Dry matter, crude protein, crude fat, crude ash, calcium, and phosphorus were

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determined in the feed and in the ileal digesta according to standard laboratory methods.16

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Amino acids in the diets and in the ileal digesta were measured by ion exchange

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chromatography (Biochrom 20 Plus, Amersham Pharmacia Biotech, Freiburg, Germany)

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following standard procedures after acid hydrolysis in 6 M HCl for 22 h ([EC] 152/2009).

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Methionine and cysteine were measured after oxidation (H2O2/formic acid).

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Titanium concentration in all experimental diets and in ileal digesta was determined by atomic

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absorption spectrometry (AAS vario 6, Analytik Jena AG, Jena, Germany) as previously

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described.18 The apparent ileal digestibility (AID) of the nutrients was calculated using

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standard equations.19

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RNA Extraction and Microarray Analysis

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Liver and jejunum samples of 4 birds per treatment group were selected for the preparation of

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total RNA. In brief: RNA was extracted using Tri reagent (Invitrogen, Karlsruhe, Germany),

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followed by DNaseI treatment (Roche, Mannheim, Germany), and column based RNA

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purification (NucleoSpin RNAII, Macherey-Nagel, Düren, Germany) according to

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manufacturer’s protocols. Quantity, integrity, and purity of RNA were checked as previously

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described.20 Subsequently, liver and jejunum total RNA samples were prepared for

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hybridization to Chicken GeneChip Gene 1.0 ST arrays using the WT Plus Expression Kit 7 ACS Paragon Plus Environment


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(both Affymetrix, Santa Clara, CA, USA). After hybridization, staining and scanning, raw

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data were generated with Affymetrix GCOS 1.1.1 software (MIAME) and deposited in a

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MIAME-compliant database, the National Center for Biotechnology Information Gene

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Expression Omnibus (www.ncbi.nlm.nih.gov/geo) (accession numbers: GSE95568). The

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Robust Multichip Average approach was employed to normalize raw intensity data. Less

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abundant probe sets and those that were detected at relatively constant levels across all

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samples were removed in order to improve statistical power. Accordingly, probe sets with

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variances less than the 50% quantile of all values and small means (log-transformed means

Possible molecular mechanisms by which an essential oil blend from

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