**1. Introduction**

Parasitic infection in ruminants, mainly by gastrointestinal nematodes (GINs), influences the intensity of emissions of greenhouse gases and substantially increases the yield of methane emission compared to uninfected animals [1,2]. Promising new nutraceuticals containing bioactive components in ruminant nutrition, however, could have both anthelmintic and anti-methanogenic properties [3,4]. Plant additives with bioactive components can modulate the bacterial, archaeal, and eukaryotic populations in the rumen by interactions

**Citation:** Petriˇc, D.; Komáromyová, M.; Batt'ányi, D.; Kozłowska, M.; Filipiak, W.; Łukomska, A.; Slusarczyk, S.; Szumacher-Strabel, M.; ´ Cie´slak, A.; Várady, M.; et al. Effect of Sainfoin (*Onobrychis viciifolia*) Pellets on Rumen Microbiome and Histopathology in Lambs Exposed to Gastrointestinal Nematodes. *Agriculture* **2022**, *12*, 301. https:// doi.org/10.3390/agriculture12020301

Academic Editor: Robert Dixon

Received: 11 January 2022 Accepted: 16 February 2022 Published: 21 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

between diet and the microbes because the microbiome plays a crucial functional role in nitrogen use, fermentation, and methane concentration [5].

Plant bioactive components such as flavonoids and condensed tannins (CTs) in feeds have the potential to reduce environmental methane pollution from ruminants by complex bioactivity occurring simultaneously in plants and animals [6]. The main bioactive components in the tanniferous legume sainfoin (*Onobrychis viciifolia*) are flavonoids and CTs formed by the polymerization of flavan-3-ols, with high proportions of prodelphinidins (70%) and procyanidins (30%) [7]. Tanniferous forages are rich in prodelphinidins, have higher antiparasitic activity, and have the effect of reducing methane emissions [8–10]. Sainfoin also reduces the degradation of feed proteins without affecting the digestibility of the nonprotein fraction, thereby increasing the flow of non-ammonia nitrogen and essential amino acids into the small intestine and reducing urinary nitrogen losses [11,12]. The ability of sainfoin to reversibly bind proteins leads to a reduction in GIN parasitism in small ruminants [13,14]. Many studies have focused on the use of sainfoin for its nutritional and anthelmintic effects, but it also contains beneficial flavonoids with similar mechanisms of action as tannins and similarly interferes with the biology of GINs [15]. Combining CTs with quercetin or luteolin identified synergistic anthelmintic effects between tannins and flavonoid monomers [16]. The production of sainfoin pellets (SFPs) at high temperatures and pressure does not affect their bioactivities associated with antioxidative properties [17]. Based on previous studies [18,19], we hypothesized that SFPs would also contribute to desired changes in the ruminal microbiome and histopathology in lambs loaded with parasites.

Analyses of ruminal microbiomes and histopathological observations are needed to identify the possible consequences of bioactive components used in the nutrition of parasiteladen lambs. Our aim was to (1) identify the main flavonoids and phenolic compounds of the SFPs and (2) determine the ruminal fermentation and microbiome, hematological profile, and histopathology of the abomasum of lambs infected with GINs during consumption of SFPs for 14 d.

#### **2. Materials and Methods**

## *2.1. Ethics Statement*

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 22 November 2020 (protocol code 2020/21).

#### *2.2. Animals, Diets, and Experimental Design*

We housed 24 male lambs (Improved Valachian) 3–4 months of age with an average initial body weight of 15.0 ± 2.07 kg in common stalls for 7 d for a period of adaptation and another 7 d for acclimatizing to feeding, with free access to water. The lambs were obtained from a commercial farm (PD Ružín–Ružín farm, Kysak, Slovakia) where they were also housed during the experiment. Each animal was fed daily meadow hay (MH) *ad libitum* and 300 g dry matter (DM) Mikrop COJ, a commercial concentrate (MIKROP, ˇ Ceb ˇ ín, Czech Republic). The number of animals used in the experiment was assigned following VICH GL13 guidelines (Veterinary International Committee on Harmonization—Efficacy of anthelmintics: specific requirements for ovine). At the beginning of the experiment—day (D) 0, all parasite-free lambs were infected orally with approximately 5000 third-stage larvae of the MHCo1 strain of *Haemonchus contortus* susceptible to anthelmintics [20]. A modified McMaster technique [21] with a sensitivity of 50 eggs per gram (EPG) of faeces was used for detecting *H. contortus* eggs on D30. The lambs were divided into two groups of twelve animals each (one stall per group) on D30 after infection, when all parasites had matured to the adult stage: control animals fed MH (control, MH, 600 g DM/d/animal) and animals fed sainfoin pellets (SFPs, 600 g DM/d/animal). Both groups continued to be fed commercial concentrate (300 g DM/d/animal). All lambs were positive with a mean

EPG of 9405 ± 4584 in the SFPs group and a mean of 11420 ± 372 in the control group. SFPs were obtained from a commercial source (NATURE'S BEST, EQUOVIS GmbH, Münster, Germany). This feeding scheme continued for 14 d. The lambs were weighed at the end of the experiment and had an average final body weight of 18.3 ± 3.22 kg. All animals were killed at the end of the experiment following the rules of the European Commission (Council Regulation 1099/2009) for slaughtering procedures [22].

#### *2.3. Experiment In Vitro*

In vitro gas fermentation technique (IVFT) has been widely used to evaluate the nutritive value of feeds for ruminants and to assess the effect of different nutritional strategies on methane (CH4) production. Therefore, IVFT using batch-culture incubations of buffered ruminal fluid incubated at 39 ◦C for 24 h under anaerobic conditions was used [23]. Control animals were donor animals for control groups and SFPs animals were donor animals for SFPs groups for the in vitro experiment. At the end of the experiment the ruminal contents (RCs) were taken from each lamb of each group immediately after slaughter in the abattoir, packed in prewarmed flasks and transported to the laboratory. RCs were pushed through four layers of gauze and pooled in equal volumes based on control and SFP groups. The pooled RCs were purged with CO2, mixed with McDougall's buffer [24] in a 1:2 ratio, and dispensed in volumes of 35 mL into fermentation bottles (120 mL) containing 250 mg (DM basis) of substrate. The meadow hay or SFPs were used as the substrates of a ration with commercial concentrate (800:200, *w/w*) as the components of the diets for the controls and SFP groups for in vitro experiment. Commercial concentrate, MH, and SFPs were ground using a grinder (Molina, MIPAM, Cesk ˇ é Budˇejovice, Czech Republic) and sieved through 0.15–0.40 mm screens. The in vitro experiment had a completely randomized design using the two diets (control and SFP) in fermentations with the two inocula of ruminal fluids (control and SFPs), with three replicates (three incubation bottles) for each diet and inoculum. The in vitro experiment was repeated three times within three consecutive days (*n* = 3 × 3).

#### *2.4. Chemical Analysis of the Dietary Substrates*

The chemical compositions of the dietary substrates (Table 1) were analyzed using standard methods [25,26].


**Table 1.** Chemical compositions of the dietary substrates.

DM, dry matter; NDF, neutral detergent fiber; ADF, acidic detergent fiber; CP, crude protein; N, nitrogen; SFPs, sainfoin pellets.

#### *2.5. Analysis of Bioactive Compounds*

SFPs were ground to a fine powder, and 100 mg were extracted three times with 80% MeOH at 40 ◦C for 60 min. The extracts were evaporated to dryness and were then dissolved in 2 mL of Milli-Q water (acidified with 0.2% formic acid) and purified by solidphase extraction using an Oasis HLB 3 cc Vac Cartridge (Waters Corp., Milford, CT, USA) as was previously described [27]. Bioactive compounds were analyzed by ultrahigh resolution mass spectrometry (UHRMS) on a Dionex UltiMate 3000RS system (Thermo Scientific, Darmstadt, Germany) with a charged aerosol detector connected to a high-resolution quadrupole time-of-flight mass spectrometer (Compact, Bruker Daltonik GmbH, Bremen, Germany). Phenolic acid and flavonoids were identified chromatographically on a Kinetex C18 column (2.1 × 100 mm, 2.6 μm, Phenomenex, Torrance, CA, USA), with mobile phase A consisting of 0.1% (*v*/*v*) formic acid in water and mobile phase B consisting of 0.1% (*v*/*v*) formic acid in acetonitrile, as was previously described [19]. Stock solutions of hyperoside and chlorogenic acid were prepared in 80% MeOH at concentrations of 2.5 and 3.6 mg/mL,

respectively, and kept frozen until used. Calibration curves for these two compounds were constructed based on seven concentration points (from 500 to 3.6 μg/mL). Hyperoside was used to calculate the number of flavonoids identified in the extract, and chlorogenic acid was used for phenolic acids, using Bruker QuantAnalysis 4.3 software (Bruker Daltonik GmbH, Bremen, Germany). All analyses were performed in triplicate.

#### *2.6. Basic Ruminal Fermentation Analysis*

RC samples from the in vitro and in vivo experiments were collected for determining pH, methane, volatile fatty acids (VFAs), ammonia concentrations, in vitro DM digestibility (IVDMD), and population of ruminal microorganisms (bacteria, protozoa, and methanogens). Concentrations of methane in vitro and VFAs were determined by gas chromatography on a PerkinElmer Clarus 500 gas chromatograph (Perkin Elmer, Inc., Shelton, CT, USA) [28]. Methane concentration in vivo was calculated by measuring the molar proportions of the VFAs in the rumen as: 57.5 mol glucose = 65 mol acetate + 20 mol propionate + 15 mol butyrate + 60 mol CO2 + 35 mol CH4 + 25 mol H2O [29]. The concentration of ammonia-N was determined using the phenol-hypochlorite method [30].

### *2.7. Rumen Microbial Quantification*

Samples for counting ciliate protozoa from the RCs were fixed in equal volumes of 8% formaldehyde, and the protozoa were counted and identified microscopically [31]. Total bacteria, *Archaea*, *Methanobacteriales*, and *Methanomicrobiales* from the in vitro experiment and *Archaea* and *Methanobacteriales* from the in vivo experiment were quantified using fluorescence in situ hybridization as described previously [32]. DNA for quantifying bacteria was isolated from the ruminal samples using a Mini Bead-Beater (BioSpec, Bartlesville, OK, USA) to lyse the cells [33] followed by purification using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). DNA concentrations and qualities were measured using a NanoPhotometer R NP80 (Implen GmbH, München, Germany). Total bacteria, *Streptococcus bovis, Butyrivibrio proteoclasticus, B. fibrisolvens, Fibrobacter succinogenes, Megasphaera elsdenii, Ruminococcus albus, R. flavefaciens,* and the genera *Prevotella and Lactobacillus* were quantified by real-time PCR using the PCR primers [34–39].
