Chicory modulates the rumen environment in lambs with endoparasites

The most natural areas typically host a variety of plant species that serve as nutritional centers and natural pharmacies with primary (nutrients) and secondary (pharmaceutical) compounds. Bioactive substances of plant origin (polyphenols) may benefit ruminants by fighting gastrointestinal parasites and bacteria and improving nutrition or reproduction¹. Plants produce various plant secondary metabolites, such as phenols, alkaloids and terpenes, which are responsible for their therapeutic effect, diversity, synergy and combinations that can contribute to different pharmacological efficacies. Self-medication of grazing ruminants on meadow pastures containing mixed plant species responds to the general demand to reduce the use of synthetic chemicals in agriculture and promote organic farming systems^2,3. However, grazing ruminants are at constant risk of getting infected by larvae of parasitic gastrointestinal nematodes (GINs). In small ruminants, GIN diseases are mainly caused by the parasite Haemonchus contortus, whose adults live attached to the wall of the abomasum and feed on the host’s blood. The control of GINs is usually limited to the repeated use of anthelmintics in conjunction with grazing management strategies⁴. However, the development of GINs in the host abomasum also causes pathology with mucosal damage and gastropathy with protein loss, followed by host inflammatory immune responses^5,6. A search for suitable natural alternatives to chemotherapeutic agents that could be used for the control and therapy of GINs is therefore needed. Many studies of medicinal perennial plants or natural products against GINs have been published in the last decade^7,8. Chicory (Cichorium intybus) is a woody, perennial herbaceous plant of the family Asteraceae, and phytochemical screening of chicory confirmed the presence of tannins, saponins and flavonoids^9,10. Chicory is also considered a quality forage with high contents of some trace minerals (e.g. Mg and Zn) that can also improve the rate of growth of grazing animals^11,12. The ruminal microbiome is responsible for the nutritional and metabolic needs of ruminants, so it can be influenced by several host factors such as age, breed, disease, infection and feed^13,14. Changes in nutrient composition can also alter the composition of the ruminal microbiota and its enzymatic activity¹⁵. Therefore, analyses of ruminal microbial fermentation and morphological observations of rumen are needed to identify possible consequences of bioactive compounds used in the diet of parasite-laden lambs.

The diversity and synergy of bioactive compounds and various combinations in medicinal plants used in our previous treatments contributed to some pharmacological efficacy against H. contortus infection^16,17,18,19. These treatments can also influence antioxidant parameters in the abomasal mucosa and help trigger a local immune response in tissues²⁰. Finally, we observed that even the replacement of meadow hay with sainfoin pellets could directly affect the dynamics of infection and the composition of ruminal methanogenic bacteria after 14 days of supplementation to infected lambs^21,22. Based on these previous studies with medicinal plants in lambs, we hypothesized that grazing different plant species, especially chicory, would alter not only the dynamics of infection but also ruminal microbial fermentation properties and histology. Our goal was to determine whether meadow pasture with a mixed species diversity of plants and/or enriched with experimentally sown chicory could favorably affect growth parameters and the ruminal environment of lambs with endoparasites.

Growth parameters of the lambs

All lambs used in the experiment had similar initial body weights (BWs, Table 1). BW differed on the days (D) D89 (P = 0.029), D131 (P = 0.050) and D144 (P = 0.027) and reached higher values in the chicory (CHIC) group (28.2, 33.0, and 38.2 kg) than in the control (CON) group (24.5, 29.1, and 32.7 kg). Live weight gains (LWGs) were higher in the CHIC group (5.10 and 5.16 kg) than in the CON group (2.28 and 3.56 kg) on D89 (P = 0.004) and D144 (P = 0.017). Daily weight gain (DWG) was higher in the CHIC group (P < 0.001; 163.8 kg) than in the CON group (131.0 kg).

Ruminal fermentation characteristics

The concentration of ammonia N was higher in the CHIC group (P = 0.007; 234 mg/L) than in the CON group (170 mg/L) (Table 2). The molar proportions of propionate (P = 0.016) and n-caproate (P = 0.049) were higher in the CON group (14.4 and 0.08 mol%) than in the CHIC group (11.7 and 0.031 mol%). Other fermentation parameters did not differ significantly between the groups (P > 0.05). The specific enzymatic activities of amylase (P = 0.008) and xylanase (P = 0.049) were significantly higher in the CHIC group (1.85 and 73.6 µcat/g protein) than in the CON group (0.94 and 54.2 µcat/g protein), but the activity of CM-cellulase was unaffected (P > 0.05). The total protozoan population did not differ significantly between the groups (P = 0.167).

Ruminal bacterial population

Total bacterial populations and the relative abundances of Streptococcus bovis were significantly higher (P < 0.05) in the CHIC group than in the CON group (Fig. 1). The other microbial populations, such as those of Butyrivibrio proteoclasticus, B. fibrisolvens, Fibrobacter succinogenes, Prevotella spp., Ruminococcus albus, R. flavefaciens and methanogens did not differ significantly (P > 0.05) among the groups.

Ruminal histology

The homogeneity of the papillae was affected in both the CON and CHIC groups. The lamina propria was inflamed in the CON group in 75% of cases and the CHIC group in 50% of cases, and the epithelia were inflamed in both groups in 25% of cases. Connective-tissue edema was present in the majority of lambs from the CON and CHIC groups, but hyperemia was present only in the CHIC group in 12.5% of cases. The epithelium of the CON group was moderately wide, with a thick layer of desquamation and balloon-shaped keratinocytes and organisms with Balantidium coli morphology were present (Fig. 2a). Hydropic degeneration of epithelial cells, hyperkeratosis, lymphocytic aggregates, connective-tissue edema and protozoa were present in the CON group (Fig. 2b and c). There were balloon-shaped keratinocytes under the rough layer, connective-tissue edema, and inflammation in the lamina propria of the epithelium in the CHIC group (Fig. 3a). The loss of ruminal papillae architecture due to massive neutrophilic inflammation with parakeratosis and the ballooning of keratinocytes in the CHIC group is shown in Fig. 3b. Connective-tissue edema, infiltration of inflammatory cells, hyperemia and extravasation in the CHIC group are shown in Fig. 3c.

Infection caused by GINs can have a strong negative impact on the productivity of sheep flocks associated with reduced rates of growth or body weights⁴. Our previous results indicated that BW or LWG may¹⁶ or may not be affected by GIN infection²³, consistent with the majority of studies in the meta-analysis reporting a negative effect of parasitism on growth and production. These effects, however, were significant in only 58.3% of the studies²⁴. Our results indicated that before D62 post-infection, BWG and LWG were comparable in both groups of lambs, which was consistent with the majority of our trials with medicinal plants against GINs in the diets of infected lambs conducted indoors^17,19,25. In the present experiment, the lambs grazing on pasture were probably able to seek out plants with certain medicinal, antioxidant or immunological effects and could also increase the consumption of plants containing secondary compounds with antiparasitic effects as self-medication against GINs^3,26. A possible loss of appetite in subclinically infected ruminants can lead to a decrease in growth performance (BW and LWG)^27,28,29. Furthermore, some byproducts of infection may likely affect the taste of infected animals^3,26. However, the growth of the lambs in the CHIC group increased significantly after D89 post-infection and growth performance expressed as DWG was significantly higher (131.0 vs. 163.8 g/day, Table 1). The meadow grasslands enriched with experimentally sown chicory were likely more beneficial for improving lamb growth³⁰ than the meadow grassland in the control plot.

This finding was in contrast to previous results³¹, which reported no effect of including chicory in the diets of grazing beef bulls on their performance compared to beef bulls grazing on ryegrass. Moreover, the levels of phytochemicals on both pastures (Tables 3 and 4) were higher in May and July compared to September, along with a lower diversity of analyzed phytochemicals in September. Better growth results were nevertheless achieved in the CHIC group. Some plants, e.g., Artemisia absinthium³², or the herbal mix composed of plant parts from Solanum xanthocarpum, Hedychium spicatum, Curcuma longa, Piper longum and Ocimum sanctum³³ improve feed consumption due to their bioactive compounds and can also possibly increase the rate of growth of lambs. On the other hand, chicory phytochemicals have properties that improve the welfare of animals with endoparasites^9,34. Therefore, the significant results for growth performance in the CHIC group during the last 55 days of the experiment were not surprising, and the differences in BW and LWG of the grazing lambs between the CHIC and CON groups in favor of the CHIC group confirm the value of feeding chicory³⁵. The self-medication of grazing lambs improved the nutrition and well-being of the animals, but the meadow pastures with experimentally sown chicory increased the efficiency of nutrient use and improved the production indicators of the infected lambs. Infected grazing lambs could choose the medicinal plant themselves while having a variety of nutritional alternatives available to them.

The concentrations of ruminal ammonia-N and microbial protein were not only affected by the intake of dietary protein and energy but also by microbial metabolism. The concentration of ruminal ammonia-N in the present study was higher in the CHIC group than in the CON group. However, the concentration of ammonia-N in the rumen can influence the degradation of fiber, so each feed has an optimal ammonia concentration³⁶, which increases immediately after feeding for 2 to 3 h and decreases until the next feeding³⁷. The capacity of protein synthesis and the use of ammonia-N in our experiment depended on the rate of carbohydrate fermentation, and presumably, greater fermentation in the CHIC group determined higher feed efficiency and allowed for elevated ammonia-N levels. Therefore, the higher ammonia-N concentrations in this study were attributed to the lower neutral-detergent fiber and acidic-detergent fiber contents (Table 2) in the pasture with chicory compared to the control pasture³⁸. In general, if energy and ammonia-N concentrations are not synchronized in the rumen, ammonia-N concentrations are not constant in the rumen^39,40. According to the literature, the optimal level of ammonia-N in the rumen varies (50 − 80 mg/L⁴¹, 50 − 190 mg/L⁴², or 60 − 290 mg/L⁴³ and depends on the optimal ammonia-N concentration for the maximal rate of fermentation⁴⁴ and the maximal production of microbial proteins^41,42. Feed intake and nutrient supply in the CON and CHIC lambs depended mainly on the rate of fermentation, and the ammonia-N concentration was probably optimal for the maximum rate of digestion. Chicory can modulate ruminal fermentation to reduce ammonia-N concentration⁴⁵. The lower ammonia-N concentrations in the CON group compared to the CHIC group could also be due to higher ammonia-N uptake by ruminal microorganisms or to lower proteolytic activity in the rumen, but the concentrations were consistent with the content of crude protein of the feed substrates from the control and experimental plots (Table 5).

The ruminal functions of lambs are usually altered in response to H. contortus infection, and infected sheep have lower ruminal propionate concentrations⁴⁶. On the other hand, a mixed infection with H. contortus and Trichostrongylus colubriformis in sheep shifted short-chain fatty acids (SCFA) production toward the propionate proportion⁴⁶. However, the molar proportion of propionate in the CON group was similar to that in our previous experiment with lambs on medicinal herbals, but without H. contortus infection⁴⁷. The higher propionate concentration in the CON group compared to the CHIC group in this experiment could indicate that more energy and nutrition were available to support the growth in the CON group when the SCFAs were absorbed and converted to nutrients in the rumen⁴⁸. However, only the propionate concentration was higher in the CON group, which could also indicate that propionate formation competed with methanogenesis for metabolic hydrogen in the rumen⁴⁹. Although the relative abundances of methanogens did not differ significantly between groups (Fig. 1), the ruminal microbiota likely promoted propionate formation in the CON group and partially inhibited methanogenesis to mitigate methane emissions⁵⁰.

In addition to increasing the abundance of bacterial genera associated with methanogenesis, GIN infection can also increase the abundance of other bacterial genera involved in microbial homeostasis⁵¹. Bioactive plant compounds from Acacia mearnsii can also modulate the microbiome in the rumens of lambs with GIN infections (e.g. affect bacterial counts and N metabolism)⁵², which is consistent with our results. Thus, the ruminal microbiome could have been affected by higher ammonia-N concentrations in the GIN-infected lambs, affecting microorganisms and the genes controlling metabolic pathways involved in microbial homeostasis, and by bioactive compounds present in the pastures. On the other hand, no effects on nutrient metabolism and methane emissions were detected during the relatively short 36-day experiment in sheep⁵³, despite the significant effects of GIN infection on animal health. The lambs in the CHIC group may have adapted to chicory grazing with an increased shift in the relative abundances of total ruminal bacteria and amylolytic bacteria (S. bovis). This shift was also accompanied by the increased specific activity of α-amylase, which is usually associated with the particulate fraction⁵⁴. However, protein metabolism during GIN infection is affected more by infection than fiber metabolism⁵⁵. Therefore, GIN infection probably increased carbohydrate metabolism in the rumen based on higher α-amylase activity in the CHIC group, compensating for the loss of the supply of energy from depressed protein and lipid metabolism caused by infection²⁹. The ruminal microbiota of the CHIC group was probably more abundant than the CON group, also associated with xylanase activities, which accelerate the biodegradation of xylan into SCFAs and gases during the ruminal processing of lignocellulosic biomass⁵⁶. However, some bacterial phylotypes can also contribute to differences in feed efficiency and host productivity and can, or need not, depend on the diet⁵⁷.

The ruminal papillae in almost all lambs of both groups were long or moderately long and thin, with mechanical degradation of papillary homogeneity. SCFAs, as products of ruminal fermentation and especially butyrate, stimulate the development of ruminal papillae and induce morphofunctional changes^58,59. SCFAs in the rumen are absorbed through the ruminal epithelium, and the rate of absorption is primarily influenced by their concentration, the surface area of the papillae in the rumen and the availability of transport proteins^60,61. Fermentation was greater in the CHIC group, which determined the higher feed efficiency, even though the molar proportion of butyrate was not affected in our experiment. Evidence supports a more active gastrointestinal tract associated with improved feed efficiency and genes associated with feed efficiency, supporting relationships between metabolic activity and ruminal epithelial function and structure⁶². However, almost all lambs had thin epithelia at D144 post-infection, indicating lower metabolic and functional activity in the papillary epithelium⁶³.

Thus, the growth and development of the ruminal papillae probably depended mainly on the type of feed consumed, and their development was stimulated by the final products of ruminal fermentation and by the composition of the pasture. However, the connective-tissue edema, inflammation of the lamina propria with infiltrates of inflammatory cells, especially lymphocytes, and the presence of singular organisms with the morphology of B. coli were detected in all lambs of both groups. The damage was probably caused by dystrophic epithelial changes that led to cellular degeneration and the infiltration of leukocytes. These lesions may affect absorptive capacity and stimulate inflammation and secondary infection of the rumen by the resident microbial population⁶⁴, which was consistent with our results.

Control and experimental plots

The experiment was carried out on pastures on a private farm (PETLAMB) in Petrovce, district of Prešov, Slovakia. Two plots with an area of 0.43 ha of meadow grassland with a mixed species of plants were selected and fenced with electric fencing. The plots were equipped with an automatic water trough and a shelter. The control plot consisted exclusively of meadow grassland with plants from different families, which were identified based on the atlas of plants⁶⁵, i.e. Astrantia major, A. carniolica, Daucus carota, Achillea millefolium, A. nobilis, Centaurea nigrescens, Cichorium intybus, Cirsium oleraceum, C. vulgare, Crepis sancta, Erigeron annuus, Hypochaeris glabra, Chamaemelum nobile, Solidago nemoralis, Tussilago farfara, Lepidium draba, Campanula patula, Knautia arvensis, Calystegia sepium, Carex hirta, Dryopteris filix-mas, Equisetum sylvaticum, Trifolium pratense, T. repens, Vicia hirsuta, V. sepium, Centaurium erythraea, Geranium maculatum, G. pratense, Hypericum perforatum, Clinopodium vulgare, Galeopsis pubescens, Mentha longifolia, Prunella vulgaris, Stachys officinalis, S. palustris, Thymus pulegioides, Lythrum salicaria, Plantago lanceolata, P. major, Veronica spicata, Agrostis capillaris, Calamagrostis arundinacea, C. epigejos, Setaria pumila, Lysimachia vulgaris, Alchemilla xanthochlora, Filipendula ulmaria, F. vulgaris, Fragaria vesca, Prunus domestica, Rosa canina, Rubus ursinus and Urtica dioica. The main polyphenolic compounds in the mixture of plants from the control plot (Pasture I—harvested May-July and Pasture II—harvested September) are shown in Table 3. The peak numbers in chromatograms represent the list of the main phytochemicals as numbered in Supplementary Tables S1 and S2. 25% of the second plot was used for reclamation with chicory. The chicory mixture contained 81.5% chicory (Cichorium intybus), 14.8% sainfoin (Onobrychis viciifolia) and 3.7% alfalfa (Medicago sativa). The main polyphenolic compounds in the chicory plot collected from May to July and from the last collection in September are shown in Table 4. The peak numbers in chromatograms represent the list of the main phytochemicals as numbered in Supplementary Table S3. The land on these plots had never been grazed by animals and was therefore considered virtually worm-free. The plots were established in the spring, and the experiment was conducted between May and October. The experiment was part of a larger study that investigated natural chemotherapeutic alternatives for controlling haemonchosis in lambs and had been described in more detail previously²⁶.

Animals, diets and experimental design

Sixteen male and female (1:1) Tsigai breed lambs aged 3–4 months with an average weight of 13.6 ± 0.52 kg from a sheep farm (PD Ružín–farm Ružín, Kysak, Slovakia) were dewormed with the recommended dose of albendazole (Albendavet 1.9% susp. a.u.v, DIVASA-FARMAVIC S.A., Barcelona, Spain) during a 7-day (D) period of adaptation before the start of the experiment. Each animal was fed 300 g dry matter (DM) Mikrop ČOJ, a commercial concentrate (MIKROP, Čebín, Czech Republic). The animals were randomly divided into two groups of eight lambs each (n = 8/group) based on their live weights and genders: control animals grazing on the control plot (CON) and animals grazing on the experimental plot (CHIC). The number of animals used in the experiment was assigned following VICH GL13 guidelines proposed by the European Medicines Agency. All parasite-free lambs were orally infected with approximately 5000 third-stage larvae of the MHCo1 strain of H. contortus susceptible to anthelmintics. The CHIC group started to graze the chicory-enriched pasture on D34 post-infection when the infection peaked. Fecal samples were collected rectally on D21, D34, D48, D62, D76, D89, D103, D118, D131 and D144 post-infection, and the number of eggs per gram (EPG) of feces was quantified. A modified McMaster technique⁶⁶ with a sensitivity of 50 EPG was used for detecting H. contortus eggs. All lambs were positive, with the highest mean EPGs of 12 800 ± 4293 in the CON group and 19 750 ± 8744 in the CHIC group on D34 post-infection²⁶. The lambs were weighed on D7, D34, D62, D89, D131 and D144. BW, LWG and DWG were examined during the experiment. The experimental period was 144 d from May to October, and the lambs were euthanized following the rules of the European Commission⁶⁷. The carcasses were sent to the Department of Pathological Anatomy and Pathological Physiology, University of Veterinary Medicine and Pharmacy in Košice in the Slovak Republic. The rumens were taken from each lamb in each group immediately after slaughter in the abattoir and transported to the laboratory.

Ethics statement

We confirm that all methods were performed in accordance with the relevant guidelines and regulations. The study was conducted following the guidelines of the Declaration of Helsinki and national legislation in the Slovak Republic (G.R. 377/2012; Law 39/2007) for the care and use of research animals. The experimental protocol was approved by the Ethical Committee of the Institute of Parasitology of the Slovak Academy of Sciences on January 20, 2023 (protocol code 14/2023). All sheep were euthanized using an overdose of 140 mg/kg pentobarbital (Dolethal, Vetoquinol, Ltd., Towcester, UK) at the end of the experiment at the abattoir (abattoir of the Centre of Biosciences of SAS, Institute of Animal Physiology, Košice, Slovakia, No. SK U 03023). The pentobarbital overdose had a negligible effect on the estimated parameters in the lambs. The study is reported following ARRIVE guidelines.

Measurements and chemical analysis

Samples from an area of 0.2 × 0.2 m were collected from 10 random quadrats in the control and chicory pastures from May to September. These samples and commercial concentrate were analyzed in triplicate by standard procedures^68,69. The chemical compositions of the feed are given in Table 5.

Analysis of polyphenols by UPLC-QTOF-MS

The polyphenols were analysed as was previously described²⁶. DataAnalysis 4.3 software (Bruker Daltonics GmbH, Bremen, Germany) provided a ranking according to the best fit of measured and theoretical isotopic patterns within a specific mass accuracy window. The quality of the isotopic fit was expressed by the mSigma-value. From Smart Formula 3D matched peaks were sent to the MetFrag website in silico fragmentation for computer-assisted identification of metabolite mass spectra. Additionally, a few databases were used to search for the structural identity of the metabolites supported with appropriate literature information^70,71, the human metabolome database (http://www.hmdb.ca/), the BiGG database (http://bigg.ucsd.edu/), the PubChem database (http://pubchem.ncbi.nlm.nih.gov/), the MassBank database (http://www.massbank.jp), KEGG (www.genome.jp) and the Metlin database (http://metlin.scripps.edu). The amount of the particular compounds (phenolic acids, flavonoids and anthocyanidins) in the extracts were calculated as equivalents of chlorogenic acid (CAS 327-97-9, 3-Caffeoylquinic acid) at 320 nm UV, isoquercetin (CAS 482-35-9, Quercetin 3-o-glucopyranoside) at 254 nm UV and cyanidin chloride (CAS 528-58-5) at 520 nm. Stock solutions of chlorogenic acid, isoquercetin and cyanidin were prepared in MeOH at concentrations of 3.2, 4.5 and 1 mg/mL respectively, and were kept frozen until analysis. Calibration curves for these three compounds were constructed based on seven concentration points. After data acquisition, raw UPLC-QTOF-MS spectra (negative mode) were pre-processed using ProfileAnalysis software (version 2.1, Bruker Daltonik GmbH, Germany).

Ruminal fermentation, microbial analysis and histology

The pH values were measured using a pH meter (InoLab pH Level 1, Weilheim, Germany). A liquid sample (4 mL) was collected for the subsequent analysis of ammonia N. The concentration of ammonia N was determined in the ruminal fluid by the phenol-hypochlorite method⁷². The SCFAs were analyzed by gas chromatography, as previously described¹⁶. Specific enzymatic activities of the ruminal microorganisms were determined by the preparation of a cell-free homogenate as described²³. Samples for microbial analyses were isolated using a PureLink Microbiome DNA Purification Kit Invitrogen (Thermo Fisher, Waltham, USA) according to the manufacturer’s protocol. The concentration of DNA was measured using Nanodrop 1 C (Thermo Fisher, Waltham, USA). Quantitative PCR was performed on a Roche Light Cycler 480 II (Roche Diagnostics GmbH, Mannheim, Germany) using standard curves for the absolute quantification of specific taxa and total bacteria by amplification of the 16 S subunit gene. The primers used were listed previously⁷³. Data are presented as the copy number of specific amplicons per total bacteria in the sample. Data are described as specific gene copy numbers per 16 S rRNA gene copy number ± SD. Samples for counting ciliate protozoa were fixed in equal volumes of 8% formaldehyde, and the protozoa were counted and identified microscopically as previously described⁷⁴. Histological examinations were performed similarly to those previously described⁴⁷.

Statistical analysis

Data were analyzed using an unpaired t-test (GraphPad Prism 9.2.0 (332) 2021; GraphPad Software, Inc., San Diego, USA). Results were considered significant at P < 0.05.

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Authors and Affiliations

1. Institute of Animal Physiology, Centre of Biosciences of Slovak Academy of Sciences, Šoltésovej 4-6, Košice, 040 01, Slovakia

   Daniel Petrič, Matej Leško, Klára Demčáková & Zora Váradyová

2. University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, Košice, 041 81, Slovakia

   Matej Leško & Klára Demčáková

3. Institute of Parasitology of Slovak Academy of Sciences, Hlinkova 3, Košice, 040 01, Slovakia

   Michaela Komáromyová & Marián Várady

4. Department of Pharmaceutical Biology and Biotechnology, Wroclaw Medical University, Borowska 211, Wroclaw, 50-556, Poland

   Sylwester Ślusarczyk

5. Department of Biochemistry and Immunochemistry, Wroclaw Medical University, Chałubińskiego 10, Wroclaw, 50-368, Poland

   Izabela Krauze

6. Department of Preclinical Sciences and Infectious Diseases, Poznan University of Life Sciences, Wolynska 35, Poznan, 60-637, Poland

   Anna Łukomska

7. Department of Genetics and Animal Breeding, Poznań University of Life Sciences, Wolynska 33, Poznan, 60-637, Poland

   Piotr Pawlak

8. Department of Animal Nutrition, Poznan University of Life Sciences, Wolynska 33, Poznan, 60-637, Poland

   Pola Sidoruk & Adam Cieslak

Contributions

DP, ML, KD and MK conducted the animal trials and performed the formal analyses. SS and IK performed the phytochemical analyses. AL performed the histological analyses. PP and PS performed the microbial analyses. AC reviewed the ethical application dossier and the manuscript. MV designed the study protocol, supervised the research and reviewed the manuscript. ZV analyzed and interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Marián Várady or Zora Váradyová.

Competing interests

The authors declare no competing interests.

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