Growth Performance and Ruminal Fermentation in Lambs with Endoparasites and In Vitro Effect of Medicinal Plants

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Introduction
The parasitic worms of ruminants, such as gastrointestinal nematodes (GINs), influence several factors associated with methane emissions, including feed efficiency, nutrient use, and animal production.Interest in research on gastrointestinal (GI) parasitism associated with ruminal fermentation and methanogenesis in ruminants is growing in Europe because ruminant GINs are very economically important [1].Recent studies have reported that GI parasitism in ruminants increases the emission of greenhouse gases and mostly affects the primary factors (the ruminal microbiome and the intake and composition of feed) responsible for enteric methane emissions [2][3][4].An association between infection by Haemonchus contortus and the diversity of the microbial communities of the abomasa and rumens of sheep from 7 to 50 d post-infection has been confirmed [5].
The ability of ruminants to convert polysaccharides In plant cell walls into meat and milk is due to a symbiotic association of a complex microbial community (bacteria, methanogenic archaea, anaerobic fungi, and protozoa) in the rumen.These microbial communities produce various enzymes that convert ingested feed into SCFAs and microbial proteins that the animal uses for growth.Due to its great diversity, the ruminal microbiome is responsible for supplying ruminants with their dietary and metabolic needs [6].Ruminal microbial communities, however, can be influenced by several host factors under specific conditions, such as age, breed, disease, infection, feed, and additives [7].GIN infections can lead to substantial changes in the digestive tract that disrupt interactions between hosts and their gut microbiomes because products secreted by GINs affect the growth and metabolism of the resident microbial communities [8,9].Changes in the composition of nutrients can also alter the composition of the ruminal microflora and its enzymatic activity [10].Plant bioactive compounds can have antimicrobial, antiparasitic, anti-inflammatory, antioxidant, and immunological properties, and their diversity, synergy, and various combinations may contribute to pharmacological efficacy in GIN infections [11,12].
Based on previous studies [13][14][15], we hypothesized that the traditional medicinal plants mallow, chamomile, fumitory, wormwood (Herbmix), and chicory would also contribute to desired changes to the in vitro ruminal microbial fermentation of GIN-infected lambs.The use of mallow, chamomile, Jacob's ladder, wormwood, and chicory in the feed of ruminants provides high-quality roughage with a high content of crude proteins (from 140 to 160 g/kg dry matter), which increases the weight gain of lambs [16][17][18][19].However, analyses of ruminal microbial fermentation in vitro are needed to identify the possible consequences of plant bioactive components used in the nutrition of parasite-laden lambs.Therefore, in this study, we performed two separate experiments (in vivo and in vitro) related to endoparasitosis in lambs.Our objectives were to (1) determine the growth performance, ruminal fermentation, and microbial population associated with GIN-infected lambs, and (2) determine the in vitro fermentation of Herbmix and chicory as substrates fermented with inoculum from GIN-infected lambs.

Ethical Study
This 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 16 February 2022 (protocol code 2022/09).

Animals, Diets, and Experimental Design
Twelve female Improved Valachian lambs, 3-4 months of age with an average initial body weight of 17.7 ± 2.12 kg, were housed together in common stalls on a sheep farm (PD Ružín-Ružín farm, Kysak, Slovakia) with free access to water.The animals were dewormed with the recommended dose of albendazole (Albendavet 1.9% susp.a.u.v,Divasa-Farmavic S.A., Barcelona, Spain) 10 d before the start of the trial and were kept indoors to maintain parasite-free conditions.Each animal was fed daily meadow hay (MH) ad libitum and 300 g dry matter (DM) of Mikrop ČOJ, a commercial concentrate (Mikrop, Čebín, Czech Republic).The twelve lambs were divided into two groups of six animals each (one stall per group): control (CON) and infected (INF).Adequate access to water and feeder space was provided for each animal.At the beginning of the experiment, six parasite-free lambs were infected orally with approximately 5000 third-stage larvae derived from the MHCo1 strain of H. contortus susceptible to anthelmintics [20].The nomenclature used in this manuscript is from the system used at the Moredun Research Institute, United Kingdom.A modified McMaster technique [21] with a sensitivity of 50 eggs per gram (EPG) of feces was used to detect H. contortus eggs six weeks after experimental infection.Two lambs per group were killed on days 48, 49, and 50 after infection following the rules of the European Commission (Council Regulation 1099/2009) for slaughtering procedures [22].
The fresh ruminal contents were collected at a slaughterhouse before the morning feeding and immediately transported to the laboratory in a 39 • C preheated water bath.

Ruminal Fermentation and Microbial Quantification
Samples of ruminal contents from the CON and INF lambs were collected for determining pH and ammonia-N concentration and identifying and quantifying the SCFAs and the populations of ruminal microorganisms.The concentration of ammonia-N was determined using the phenol-hypochlorite method [23].Samples for counting ciliate protozoa from the ruminal fluid were fixed in equal volumes of 8% formaldehyde, and the protozoa were counted and identified microscopically [24].Bacteria, total Archaea, Methanobacteriales, and Methanomicrobiales in the ruminal contents were quantified using fluorescence in situ hybridization [25].

Specific Enzymatic Activities
The specific enzymatic activities of the ruminal microorganisms were determined by the preparation of a cell-free homogenate.The ruminal content, which was immediately frozen at −80 • C after collection, was thawed before homogenization and diluted in an amount of approximately 6 g with 2 mL of a phosphate-citrate buffer solution (pH 6.8) with cOmplete TM mini EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics GmbH, Mannheim, Germany).The contents were homogenized in an ice bath using a 4710 series ultrasonic homogenizer (Merck KGaA, Darmstadt, Germany) at a power of 80 W pulsed in one-minute intervals (homogenization and cooling).All samples were centrifuged at 16,000× g for 15 min, and the supernatant was used for the enzymatic assays.The specific enzymatic activities of amylase, cellulase, and xylanase were determined colorimetrically by measuring the amount of reducing sugars released using dinitrosalicylic acid (DNSA) [26].Tubes containing 1.8 mL of substrate were placed in a thermostat at 39 • C for 5 min.One hundred microliters of the homogenate were then added to the tubes (50 µL for xylanase), and the contents of the tubes were supplemented with 100 µL (150 µL for xylanase) of a phosphate-citrate buffer solution to a volume of 200 µL.The mixtures were boiled in 3 mL of DNSA (1% solution) for 5 min to stop the reaction.The blank was prepared similarly to the experimental samples using deionized water instead of the homogenate.Standards were prepared from standard solutions and a phosphate-citrate buffer with final volumes of 200 µL.All samples, standards, and enzymatic and reaction blanks were prepared in duplicate and boiled for 5 min, and cooled, and 250 µL were transferred to a 96-well plate.Absorbances were spectrophotometrically measured against the reaction blank at a wavelength of 540 nm [27].Enzymatic activity was expressed in units of specific catalytic activity (cat/g of protein).Amylase activity was determined using 0.2% (w/v) maize starch (Merck KGaA, Darmstadt, Germany) resuspended in 0.05 M phosphate-citrate buffer.Cellulase activity was determined using 1% (w/v) carboxymethyl cellulose (Merck KGaA, Darmstadt, Germany).Xylanase activity was determined using 1% (w/v) Beechwood xylan (Merck KGaA, Darmstadt, Germany) resuspended in the same phosphate-citrate buffer.Enzymatic activities were determined by measuring the amount of reducing sugars released from the samples after 15 min at 39 • C.

Experiment 2-In Vitro
Ruminal contents were separately collected from the slaughtered CON and INF lambs.Ruminal contents were separately passed through four layers of gauze, mixed at a 1:2 ratio with McDougall's buffer [28], and dispensed in volumes of 35 mL into serum bottles containing 0.25 g of a substrate.Meadow hay (MH), a dry herbal mixture (Herbmix), and dry chicory (Cichorium intybus L.) were used as substrates.Herbmix contains flowers of mallow (Malva sylvestris L.) and chamomile (Matricaria chamomilla L.) and stems of fumitory (Fumaria officinalis L.) and wormwood (Artemisia absinthium L.) mixed in equal proportions.The plants were obtained from a commercial source (Agrokarpaty, Plavnica, Slovak Republic) and ground through a 0.15-0.40mm screen using a Molina grinder (Mipam bio s.r.o., České Budějovice, Czech Republic) or a stand mixer (Bosch, Berlin, Germany).The experiment was carried out using the in vitro gas production technique (IVGPT) on batch culture incubations of buffered ruminal fluid incubated for 24 h at 39 • C under anaerobic conditions.The volume of accumulated gas released from the recorded pressure, or the volume of gas produced after 24 h of fermentation, was determined using IVGPT [29].Gas samples (1000 µL) were collected from the headspace of the bottles using a gastight syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) for measuring the methane concentration in the samples.The SCFAs and methane in vitro were analyzed on a Perkin Elmer Clarus 500 gas chromatograph (Perkin Elmer, Shelton, WA, USA) [30].Abomasal contents were collected from the slaughtered INF lambs.Clumps of adult worms were manually collected from the abomasal contents of infected lambs, and approximately 20-30 adult worms were placed into the INF serum bottles.

Experimental Design
Three replicates (three incubation serum bottles) were prepared for MH, Herbmix, or chicory, and each inoculum (CON and INF).The experiment consisted of fermentations of the three substrates in fermentations with two inocula (CON and INF) and repeated three times within three consecutive days (n = 3 × 3).At the same time, three replicate bottles were also used for the blank (inoculum, no substrate).

Chemical Analysis of Dietary Substrates
The chemical compositions, phytochemical substances, and FA profiles of the dietary substrates used in both the in vivo and in vitro experiments are presented in Table 1.

Diet Composition
The dietary substrates were analyzed in triplicate using standard procedures [31,32] as previously described [33].The analyses were performed using an ANKOM 2000 Automated Fiber Analyzer (Ankom Technology, Macedon, NY, USA) and a FLASH 4000 N/Protein Analyzer (Thermo Fisher Scientific, Cambridge, UK).

Phytochemical Substances
The bioactive compounds in Herbmix and chicory were quantified using ultrahighresolution mass spectrometry on a Dionex UltiMate 3000RS system (Thermo Scientific, Darmstadt, Germany) with a charged aerosol detector connected to a Compact highresolution quadrupole time-of-flight mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) as previously described [20,34].

Fatty Acid Profiles
The fatty acid (FA) concentrations were determined using standard protocols [35].FAs were identified and quantified based on their peaks and retention times by comparing FA sample targets with appropriate FA methyl ester standards (Supelco 37 Component FAME Mix) (Sigma-Aldrich, Saint-Louis, MO, USA).The concentrations of conjugated linoleic acid (CLA) were determined using a CLA standard (a mixture of cis 9, trans 11 and trans 10, cis 12-octadecadienoic acid methyl esters; Sigma-Aldrich) on a Galaxie Work Station 10.1 (Varian, Walnut Creek, CA, USA).

Statistical Analysis
Fermentation parameters and microbial populations in vivo were analyzed using an unpaired t-test (GraphPad Prism 9.2.0 (332) 2021; GraphPad Software, Inc., San Diego, CA, USA).Data on fermentation parameters and protozoan populations in vitro were analyzed by two-way ANOVA (GraphPad Prism 9.2.0 (332) 2021; GraphPad Software, Inc., San Diego, CA, USA).The model included effects for three substrates (MH, Herbmix, and chicory), two inocula (CON and INF), and interactions between substrates and inocula.Individual differences were determined using Tukey's multiple-comparison post-test and were considered to be significant at p < 0.05.

Ruminal Fermentation In Vivo
Infection significantly affected ammonia-N concentration in the lambs, with a higher value for INF than CON (Table 3).The concentrations of n-butyrate (p = 0.010), iso-butyrate (p = 0.018), and iso-valerate (p = 0.003) were significantly lower, but the acetate: propionate ratio (p = 0.022) was significantly higher for INF than CON.

Experiment 2
The effects of the inocula and substrates on the parameters of ruminal fermentation in vitro are presented in Table 5.The substrates significantly affected the concentrations of n-butyrate (p = 0.048), iso-butyrate (p < 0.001), n-valerate (p < 0.001), iso-valerate (p < 0.001), ammonia-N (p = 0.021), total gas (p = 0.001), and methane (p < 0.001).The concentrations of iso-valerate were higher in CON groups than in INF groups.Ammonia-N concentration was higher in INF groups compared to CON groups.TGP did not differ between groups with the same substrates.The concentration of methane was highest in the INF group with Herbmix.The inocula significantly affected the concentrations of n-butyrate (p = 0.002), ammonia-N (p = 0.004), and methane (p < 0.001).The inocula also significantly affected the total number of protozoa (p = 0.029).The inoculum × substrate interaction significantly affected only the concentrations of n-butyrate (p = 0.002), n-valerate (p = 0.013), and methane (p < 0.001).

Discussion
Many studies have confirmed the negative impact of GIN infections on sheep performance [36][37][38].GIN infections can reduce the voluntary feed intake in ruminants, even in the absence of any clinical disease [39].In Experiment 1, growth performance expressed as DWG was relatively lower in GIN-infected lambs compared to control, but not significantly (72.6 vs. 130.1 g/day, Table 2).The results of a meta-analysis indicated that weight gain in animals infected with H. contortus was 77% of the gain in parasite-free animals [38].Most of the trials in the meta-analysis reported a negative effect of parasitism on growth performance and production, but the effect was significant in only 58.3% of the trials [38].We thus also expected worse general health conditions in the infected lambs, manifested by lower BWs 50 d post-infection.Our experiment, however, did not confirm the effect of infection on BW, probably because feeding the lambs with quality forage ensured that the lambs were free from adverse effects on animal performance.In the present experiment, large individual differences between animals were also noted when we weighed animals.It has been documented in the past that parasitic infection with H. contortus can often result in a period of reduced appetite, nausea, gastroenteritis, anemia, and poor nutritional absorption, which can lead to weight loss [40].The lambs in our experiment maintained a good nutritional status (nutritional management) with the forage within medium-to high-quality standards [41], which was evident from the optimal chemical composition of the dietary substrates (Table 1).The ability of ruminants to resist the negative impact of GIN infections when maintaining good nutritional status (nutritional management) has previously been reported [42,43].
Changes in the ruminal ammonia-N concentration in INF in our Experiment 1 may have been associated with alteration of the ruminal microbiota.The optimal level of ammonia-N in the rumen (20-100 mg/L; [44]) was exceeded in our experiment in INF.Ruminal ammonia-N is normally the most abundant nitrogenous compound needed for microbial growth; its increase in the rumens of the infected lambs may have been due to the lower consumption of ammonia-N by microorganisms.These microorganisms have access to a readily available source of energy, increasing microbial protein synthesis or decreasing the use of amino acids as a microbial energy source [45].A large increase in the amount of additional endogenous nitrogen entering the duodenum in sheep infected with H. contortus would probably lead to the loss of amino acids because the reabsorbed N not from ammonia would likely be used inefficiently [46].
In Experiment 1, concentrations of branched-chain FAs (BCFAs, i.e., iso-butyrate and iso-valerate) were significantly lower for INF than CON lambs (Table 3).BCFAs are formed in the rumen by the deamination of amino acids, and their concentrations depend on the ruminal degradation of dietary proteins [47].The relative proportion of proteins in the abomasum obtained from feed and microbial proteins synthesized in the rumen, however, depended on the physiological status of lambs and diet composition.
Our results (Experiment 1) indicated increased protozoan, bacterial, and methanogenic populations (i.e., Archaea, Methanobacteriales, and Methanomicrobiales) in GIN-infected lambs (Table 4).Infection with the GIN H. contortus altered the composition and diversity of the microbial community, which likely facilitated bacterial survival and reproduction.An increase in the abundance of bacterial genera associated with methanogenesis, and other ruminal microorganisms involved in microbial homeostasis, may be affected by infection [48,49].The ruminal microbiome may therefore be affected by decreasing butyrate and increasing ammonia-N concentrations in GIN-infected lambs, which would affect microorganisms and the genes controlling the metabolic pathway involved in microbial homeostasis.The high ammonia-N concentrations in GIN-infected lambs would consequently inhibit acetoclastic methanogens much more than hydrogenotrophic methanogens, so methane would mainly be formed by hydrogen-using methanogens [50,51].Acetate oxidation associated with hydrogenotrophic methanogenesis is probably the dominant metabolic pathway for methane formation in GIN-infected animals [52].However, changes in the ruminal microbiota, including ciliates that are responsible for methanogenesis in GIN-infected animals and determine how hydrogen is used in the rumen, remain poorly understood.The intake and composition of feed and the ruminal microbiome, however, are the primary factors influencing enteric methane emissions.
In Experiment 1, GIN infection also affected the specific activities of enzymes of the ruminal microorganisms, especially amylase (Table 4), which is associated with the particulate fraction [53].Xylanase was higher (not significantly) in GIN-infected lambs because rumen protozoa, fungi, and bacteria are associated with xylanase activities that accelerate the biodegradation of xylan to SCFAs and gases during the processing of lignocellulosic biomass in the rumen [54].Diet in our study, however, may also have contributed to the increase in amylase activity in the ruminal microorganisms in CON.
In Experiment 2, the dietary substrates containing Herbmix (a mixture of mallow, chamomile, fumitory, and wormwood) [34] or chicory [55] have the potential to reduce enteric methane and ammonia-N concentrations in vitro.Our study found differences between the in vitro fermentation of the same substrates using inocula from GIN-infected and uninfected animals.The INF inocula had lower n-butyrate and higher ammonia-N and methane concentrations (Table 5).Some fermentation parameters (total gas production, SCFA concentrations, and protozoan activity) differed significantly in the resistance of substrates to degradation in the ruminal fluid, consistent with previous results in vitro for estimating ruminal protein degradability [56].Significantly affected iso-butyrate and iso-valerate values by substrates without the effect of inocula were probably due to the high protein content of Herbmix and chicory [57,58].In addition, the ruminal cellulolytic microorganisms could also primarily use BCFAs (i.e., iso-butyrate and iso-valerate) as the main source of carbon chains for growth.
Diets with a high chicory content (≥70% DM) can directly affect GIN parasitism [13], but some studies have also described indirect anthelmintic effects of chicory and herbal mixtures on local abomasal immunities [14,59,60].Our in vitro fermentation with chicory also provided promising results in mitigating methane production, and chicory likely modulated the ruminal fermentation and microbiota of the GIN-infected lambs.Chicory was a source of coumarins in our experiment, and coumarin-rich raw materials can inhibit acetogenesis and acetoclastic methanogenesis when the microbiome is not adapted to coumarins [61].Our previous results indicated that fermentation in the rumen (in vivo and in vitro) could be modified by supplementing medicinal plants, without adverse effects on the parameters of fermentation in the rumen [28,33].The high potential of tannins and flavonoids to reduce methane and ammonia-N concentrations during ruminal fermentation in vitro has also been previously described [62][63][64].These bioactive compounds probably have similar mechanisms of action that also affect GIN larvae [65].It seems that chicory bioactive compounds may have the potential to inhibit methane production in ruminal fermentation in GIN-infected animals without adversely affecting fermentation.Antimicrobial activity varies amongst flavonoids [66], with some flavonoids enhancing the efficiency of fermentation by improving propionate production to the detriment of acetate production and reducing the hydrogenotrophic methanogenic communities of Archaea [67].Our in vitro results are consistent with either the direct effects of plant bioactive compounds on methanogens or an indirect effect on reducing the production of hydrogen as a substrate for microorganisms [68].Variations in the ruminal microbiome, fermentation kinetics, and the responses and adaptations to anti-methanogenic inhibitors and dietary substrates, however, may be important factors influencing the efficacy of bioactive compounds [69].The effectiveness of bioactive substances, though, can vary considerably depending on the type, source, molecular weight, and dose in the diet [70].

Conclusions
We did not confirm the effect of GIN infection on daily weight gain, as feeding lambs with quality feed ensured that lambs were free from adverse effects on growth performance.However, our study confirmed the effect of endoparasites on fermentation and microbial population in the rumens of the lambs.Chicory has promise for mitigating ruminal methane production in vitro and may modulate the ruminal microbial fermentation of GIN-infected lambs.However, the mechanisms and contributions of Herbmix, chicory, and gastrointestinal nematodes to the ruminal microbiome of lambs remain undefined.More in vivo studies are thus needed.

Table 1 .
Chemical composition, phytochemical substances, and fatty acid profile of the substrates.
1Mean ± standard deviation (SD).Each animal was fed daily MH ad libitum and 300 g DM commercial concentrate.