**1. Introduction**

The bioavailability of trace elements (TEs) depends on their bioaccessibility, absorption, retention, and role in animals [1,2]. Whereas bioavailability of TEs in vivo is determined by the mineral fraction, which is eventually absorbed into the systemic circulation and utilised by animals, in vitro studies define TE bioaccessibility as the fraction of minerals that is soluble in the gastrointestinal tract (GIT) and available for absorption [2,3]. Trace elements are essential for living animals to maintain their health and performance, but the generally high presence of phytate in cereal-based feedstuffs decreases the absorption of minerals from the gu<sup>t</sup> and their bioavailability [4]. In the digestive tract, divalent minerals chelate with phytic acid to form soluble and/or insoluble mineral-phytate complexes. These complexes are resistant to hydrolysis by phytase activity, and for this reason, the absorption and bioavailability of trace elements are decreased [5]. In addition, pH is an important factor influencing phytate and mineral complex solubility. During passage through the small intestine, at intestinal pH (pH 6.5) the mineral complexes become insoluble and are precipitated to decrease mineral absorption due to de novo complexation [5,6], whereby the trace minerals most affected by phytate are Zn and Fe.

Zinc as a component of many metalloenzymes performs multifarious physiological roles and is involved in almost every metabolic pathway of the body [7]. Zn compounds positively affected the growth performance, intestinal morphology, and regulation of the

**Citation:** Tokarˇcíková, K.; Cobanová, ˇ K.; Takácsová, M.; Barszcz, M.; Taciak, M.; Tu´snio, A.; Grešaková, L'. Trace Mineral Solubility and Digestibility in the Small Intestine of Piglets Are Affected by Zinc and Fibre Sources. *Agriculture* **2022**, *12*, 517. https:// doi.org/10.3390/ agriculture12040517

Academic Editor: Maria Grazia Cappai

Received: 17 December 2021 Accepted: 3 April 2022 Published: 6 April 2022

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gu<sup>t</sup> microbiota; therefore, a therapeutic dose of ZnO is used to decrease the post-weaning diarrhoea frequency in piglets [8,9]. Due to a total ban on the therapeutic use of ZnO that will be introduced from June 2022 in the European Union [10], the evaluation of different Zn sources and Zn lower doses in pig nutrition is very required and challenging. Although the allowed maximum of 150 mg Zn/kg in complete feed in the EU is an acceptable Zn level to maximize the pig performance and effectively counteract diarrhoea [11], it is necessary to improve Zn efficiency and absorption by feed composition in order to maintain animal health, welfare and productivity together with the substantial reduction of environmental pollution from animal husbandry.

Because of the different chemical properties of various Zn feed additives, solubility and interaction of TEs within the GIT may vary. Zinc sulphate together with zinc oxide are the most commonly used Zn source in pig nutrition [12]. ZnSO4 has been frequently used as a standard to compare the bioavailability of Zn from different sources [13,14], but this soluble source of Zn could more readily interact with phytates and other ligands in the GIT, resulting in lower Zn absorption [15–17]. On the other hand, different chemical structures and properties of Zn organic chelates, more stable Zn complexes, could protect Zn from interaction with other minerals and feed compounds in the gu<sup>t</sup> [2,18], thus making Zn more absorbable [13,19]. Moreover, some Zn organic sources could be absorbed from the intestine in an intact form [18]. Although phytate seems to be the most effective inhibitor of mineral absorption [15], other components such as dietary fibre, lignin, and polyphenolic compounds may bind minerals and affect their bioavailability as well [15,20]. Generally, fibre and compounds associated with fibre in cereal products reduced the mineral absorption in animals [21].

There are two general mechanisms for the inhibition of Zn absorption in the GIT, chelation precipitation (formation of insoluble complexes with phytic acid, dietary fibre, tannins) and competitive inhibition between minerals [22,23]. Feed supplementation with Zn and fibre from different sources (inorganic or organic Zn, and cellulose or potato fibre) could influence mineral absorption during the passage of feed through the small intestine. Our previous results indicated positive effects of potato fibre (PF) supplementation on nutrient digestibility [24]; therefore, we hypothesized that both dietary sources could also affect the apparent digestibility of TEs due to changes in pH and mineral solubility in the GIT. We focused on solubility and absorption of Zn and other interfering TEs in the small intestine as their main absorption site. In this study, we investigated the effect of Zn and fibre sources on the apparent digestibility of TEs (Zn, Cu, Fe, Mn) in the total digestive tract and in the small intestinal segments of piglets as well as pH in the gut. Moreover, we investigated in vitro solubility of TEs from the dietary treatments in a simulated digestion assay, and TE in situ solubility from the small intestinal digesta (duodenum, jejunum ileum) of piglets as a potential indicator of mineral bioaccessibility. Our in vitro experiment was designed to compare the influence of both dietary sources (Zn sources: ZnSO4 or ZnGly, fibre sources: cellulose or potato fibre) on mineral solubility in the whole GIT using a simulated three-step digestion assay (gastric, small and large intestinal phases).

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

All experimental protocols involving animals were performed in accordance with the Guiding Principles for the Care and Use of Research Animals and Animal Research: Reporting In Vivo Experiments (ARRIVE guidelines). All methods and procedures reported herein were carried out in line with European Union Directive 2010/63/EU for animal experiments, and the experimental protocol was approved by the Local Animal Experimentation Ethics Committee (resolution number WAW2\_21/2016, Warsaw University of Life Sciences-SGGW, Warsaw, Poland) and Polish Law on Animal Protection.
