**1. Introduction**

The use of plant extracts has increased significantly in recent years, especially after the ban on the use of nutritive antibiotics in animal production in Europe in 2006 [1]. The emergence of resistance in pathogenic microorganisms mainly originating from the digestive tract of animals has caused public concern and led breeders to reflect on the necessary changes and immediate measures in the practical conditions of breeding. The relatively positive effects of various feed supplements of plant origin, such as essential oils, tannins, and others, was quickly discovered in non-ruminant breeding, which is not identical to the effect in ruminants [2]. A digestive tract adapted to the voluminous nature of the ration, with foregut-specific microbiota, is an additional obstacle to the rapid and effortless transfer of the norms of nutritional supplements of another animal groups.

Because of the voluminous nature of the diet, cattle can already absorb many of the above-mentioned amounts of active ingredients in a varied ration. Nevertheless, in intensive beef production, it is essential to control the microbial population in the foregut and reduce opportunistic pathogenic microbes of the normal intestinal microflora. Such properties are also possessed by plant secondary metabolites and are referred to as phytochemicals, phytogenes, or phytobiotics and could be a very suitable substitute for

**Citation:** Mergeduš, A.; Janžekoviˇc, M.; Škorjanc, D.; Kraner Šumenjak, T.; Brus, M. Growth Performance, Meat Quality, and Fecal Microbial Population in Limousin Bulls Supplemented with Hydrolyzable Tannins. *Agriculture* **2022**, *12*, 939. https://doi.org/10.3390/ agriculture12070939

Academic Editors: Lubomira Gresakova, Emilio Sabia and Hao Zhang

Received: 19 February 2022 Accepted: 24 June 2022 Published: 28 June 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/).

antibiotics in animal nutrition [3–6]. Tannins from higher plant species are proving to be very promising [7].

Tannins are found in different plant species and different plant parts, including bark, fruits, leaves, and roots, resulting in different physical and chemical properties [8,9]. Therefore, the bioactive properties of tannins depend on their chemical structure, which is even more important than their concentration [10,11]. Plants produce them as secondary metabolites for their own protection against consumption by herbivores [11]. The group of tannins is very diverse and can be divided into four groups based on their chemical structure: (i) condensed tannins or proanthocyanidins; (ii) HT; (iii) florotanins from brown algae; and (iv) complex tannins conjugated with metals or proteins [12]. In the literature, they are usually classified into two broad groups: HT gallic acid and glucose and condensed tannins consisting of flavonoids [13,14], both of which are found in small amounts in forage ingredients < 450 μg g<sup>−</sup><sup>1</sup> DM, [15]. HT is divided into two subgroups, gallotannins and ellagitannins [16]. Hydrolyzable tannins are a group of water-soluble polyphenolic compounds that possess antimicrobial, anti-inflammatory, antiviral, antioxidant, and antiparasitic activities [17,18].

Feed additives according to (EC) No. 1831/2003 [19] are divided into four categories: technological additives, sensory additives, nutritional additives, and zootechnical additives. According to current knowledge, HT from sweet chestnut is classified in the second category, sensory additives, and in the subcategory (b) flavoring agents with the active substance level [20]. Therefore, our research aims to elucidate and expand the knowledge of the activity of HT beyond the bureaucratically limited framework that describes only the definition of potential effects. In the scientific and professional literature, we often find a general use of the term food supplement, which does not necessarily correspond to the definition and categorization of the EU standards for plant extracts. Some studies on tannins intended for transfer to ruminants have been performed in vitro [21], on cannulated or fistulated animals, on small ruminants, and in individual small laboratory tests. Mostly, these tests examine the effective concentration (fattening lambs—20.8 g HT kg−<sup>1</sup> DM and sheep—34.0 g HT kg−<sup>1</sup> [22], higher concentrations (generally >50 g kg−<sup>1</sup> DM [23]) on rumen microflora, mechanism and efficiency of bypass proteins [24–26], growth of animals, meat quality [18,27,28], health problems caused (kidney and liver damage [8]), and antimicrobial activity (mechanism [29] on microbes in feces (*Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Aspergillus niger* and *Candida albicans* [30], *Clostridium perfringens* [31], and *Clostridia* [30,31]). However, a variety of plant materials with unclear composition and varying tannin content have been used in research [21,23,25,32,33].

In the available literature, we found no study in which bulls were fattened under commercial fattening conditions at modest feed rations with a small amount HT of known composition. Therefore, the objective of this study was to determine the effects of adding chestnut (*Castanea sativa* Mill.) tannin extracts with a high HT content at low concentrations of 1.0 to 1.5 g kg−<sup>1</sup> DM to the diets of fattening bulls. We investigated their effect on growth and fattening traits, carcass quality and meat quality of bulls. In addition, the effect of HT on the microbiological condition (*Clostridia*) of bull feces was estimated.

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

The animal procedure was conducted at an experimental farm located in NE Slovenia (46◦5 N, 15◦8 E), followed the Slovenian Law on Animal Protection, and approved feed additives were used (European Union Register of Feed Additives, 2013) [34]. The ethics committee's approval was not required.

#### *2.1. Animals, Housing and Diets*

Thirty-two Limousin bulls were included in the 7-month trial to evaluate the effect of various levels of supplementation of hydrolyzable tannin (chestnut tannin: *C. sativa* Mill.) on growth, carcass and meat quality traits, and fecal *Clostridia* strains.

At an average body weight (BW) of 432 ± 43 kg, thirty-two bulls were randomly assigned to one of four treatment groups (a completely randomized design). The bulls were housed in collective pens (eight animals per pen), where they were kept for the duration of the trial. All identical pens located in the same barn consisted of a standard slatted concrete floor area 6 × 4 m, equipped with identical water troughs. Equal feeding conditions were ensured for all bulls and individual bulls served as experimental units.

Animal BWs were equalized at the beginning as much as possible, both within and between individual groups. Highly homogeneous groups facilitated a viable experiment and comparison of groups during the trial.

Animals were fed a total mixed ration (TMR). The composition of the basal diet is presented in Table 1. Diets were prepared daily, and rations were formulated to meet the requirements of medium-frame finishing bulls [35]. During the feeding trial, the feed ratios for bulls were optimized twice according to BW to meet their nutritional requirements.


**Table 1.** Ingredients and chemical composition of total mixed ration (g kg−<sup>1</sup> DM).

No tannin additive in the diet (CON); 10 g of mixture (HT + soy protein additive) per animal—1.0 g kg−<sup>1</sup> DM (TAN 1); 15 g of HT additive per animal—1.5 g kg−<sup>1</sup> DM (TAN 2); and 15 g of HT additive per animal added to the TMR diet with reduced quantity of concentrate in nominal value—1.5 g kg−<sup>1</sup> DM (TAN 3). Dry matter (DM); acid detergent fiber (ADF); neutral detergent fiber (NDF); acid detergent lignin (ADL); metabolizable energy (ME).

Commercially available Farmatan-D®, wood extract rich in HT, was obtained from Tanin d.d. Sevnica (Sevnica, Slovenia); the supplement originating from sweet chestnut wood (*Castanea sativa* Mill.). All wood extracts were in powder form before use. Suitable quantities of wood extracts were mixed into feed rations on the DM bases. The chemical compositions of the wood extracts rich with hydrolyzable tannins: major components (%) of Farmatan-D (hydrolyzable tannins 74.3, vescalin 0.9, castalin 1.7, roburin A 0.2, gallic acid 2.4, roburin B/C 2.1, grandinin 0.9, roburin D 1.0, vescalagin 4.7, roburin E 1.5, castalagin 4.1, ellagic acid 0.8.). The supplement was used in the three groups and compared with the control group.

The four trial groups were comprised of thirty-two animals, eight per group:

The control group (CON)—no tannin additive in the diet. The first treatment group (TAN 1)—10 g of mixture (HT + soy protein additive) per animal added to the TMR diet (1.0 g kg−<sup>1</sup> DM). The second treatment group (TAN 2)—15 g of HT additive per animal added to the TMR diet (1.5 g kg−<sup>1</sup> DM).The third treatment group (TAN 3)—15 g of HT additive per animal added to the TMR diet (1.5 g kg−<sup>1</sup> DM) with reduced quantity of concentrate in nominal value of the HT additive (Table 1).

Feed rations were prepared daily for all treatment groups. The bulls were fed their respective diets once a day (07:00 h), following a 20 d diet adaptation period. During the adaptation period, the diets were not supplemented with tannin extract. Access to feed and water was provided ad libitum. Ration components for complete feed rations, and refusal samples were collected monthly at the beginning and during fattening period, and were sent to the laboratory for chemical analyses (LKS-Landwirtschaftliche Kommunikations-, und Servicegesellschaft mbH, Germany). The mill was washed after preparation of each diet to prevent cross-contamination with tannins. The ratio was adjusted on a weekly basis. During the feeding trial, the feed rations and feed refusals were recorded on a daily basis for each treatment group. In the 7-month feeding trial, a total of 852 offered rations and 852 residual feeds were precisely measured. From the data, we calculated the dry matter intake (DMI).

#### *2.2. Recording of Dry Matter Intake*

Feed and feed refusals were collected and recorded daily for DM analysis and calculation of DMI. To calculate the monthly average DMI, the average of all daily feed and feed refusals for each pen (each group) were calculated. The results are presented as monthly average DMI per pen, as descriptive statistics.

#### *2.3. Recording of Body Weight*

Following a 20 d diet adaptation period, bulls were weighed and started the 213-day experimental period. During the experimental period, bulls were weighed at the beginning and at 30-day intervals thereafter. At the end of the experiment, the bulls were weighed 24 h before slaughter and on the day of slaughter. Body mass data were recorded using a digital walk-through scale (EC 2000 Tru-test). The average daily gain (ADG) was calculated between the monthly recordings and throughout the experiment. At each weighing, body parameters were also recorded: height at withers (measured from the highest point of the shoulder blade to the ground) and hip height (measured from the highest point of the hip bones to the ground).
