**4. Discussion**

Trace elements as essential feed components improve animal health and livestock productivity but feed supplementation with TEs result in a substantial excretion of heavy metals into the environment. Therapeutic use of ZnO or excessive feeding of inorganic Zn to piglets can stimulate resistance in the gu<sup>t</sup> microbiota, and Zn accumulates in faeces and then in manure [31]. Replacement of inorganic feed mineral additives by more bioavailable mineral sources or using enhancers of trace mineral absorption in animal feed could reduce excretion of TEs into the environment and reduce heavy metal emissions from livestock production.

Whole-body homeostasis of Zn, Mn, Cu, and Fe is predominantly regulated by intestinal absorption [2,32]. Absorption of trace elements (TEs) does not only depend on an adequate dietary intake but is also greatly affected by its intestinal availability from the diet. Different chemical species of dietary TEs can interact with other components in the diet before reaching their absorption site and these interactions may considerably modify the metabolically available amount of the trace minerals, mineral metabolism, and their regulation [16]. Sufficient Zn supply from different organic dietary sources of Zn and from inorganic ZnSO4 showed usually no differences in metabolic utilisation in animals due to Zn homeostasis adapted to intestinal Zn content [33]. However, Zn supplementation with organic ZnGly increased Zn absorption from the GIT of Zn-deficient animals [34,35]. Our results indicated the decreased apparent total tract digestibility (ATTD) of Zn in piglets fed the diet supplemented with organic Zn glycinate compared to other treatments; however, the apparent Zn digestibility did not differ in the small intestine. Higher feed consumption of piglets fed the diets supplemented with ZnGly also increased the ingested amount of Zn that could lead to increased excretion of Zn, and as a result of Zn homeostasis regulating the absorption and excretion of Zn in the GIT [32,36], Zn apparent digestibility decreased in the ZnGly treatment. However, decreased ATTD of Zn was observed in the ZnGly treatment only. It has been known that fractional Zn absorption decreases increasing Zn content in the feed, probably due to a saturation of transport mechanisms [37]. Zn absorption is more efficient in low zinc diets [38] and relates to oral zinc intake [17]; therefore, increased feed

consumption of Zn might decrease Zn uptake by the small intestine and Zn absorption. Moreover, the importance of maintaining Zn balance is endogenous Zn excretion and Zn reutilisation by GIT which directly relates to the absorbed Zn amount [39]. Another reason for the lower ATTD of Zn from ZnGly could be the structure of zinc glycinate. The small molecular size of ZnGly tends to bind in cavities of native fibre [40] resulting in the decrease of apparent Zn digestibility, but it seems that feed supplementation with PF could eliminate the effect. Reduced absorption and retention of Zn from ZnGly were observed in pigs due to the structure and lower average bond strength between the amino acid and Zn in ZnGly [40].

Although it seems that the chemical properties of trace elements are of subordinary relevance in the absorption process of dietary TEs in the GIT, their chemical speciation is relevant for complex interactions with different chelators to promote or prevent the formation of insoluble mineral complexes [22,34]. Phytates and fibres are the main ligands binding native Zn and other trace element species in the GIT [32,41] resulting in the inhibition of Zn absorption and reabsorption by chelating ingested Zn or secreted endogenous Zn in the intestinal lumen [39]. Regardless of dietary fibre source, fibres may affect mineral absorption due to their mineral binding properties [42,43] and these interactions are important from a nutritional point of view. Cellulose is a poorly soluble and fermentable fibre source that entraps minerals to form large insoluble complexes resulting in decreased mineral bioavailability; however, most of the different fibres can also bind Zn, Fe, Mn, and Cu under in vitro conditions [44]. On the other hand, more fermentable PF can enhance the absorption of TEs by lowering pH or the production of organic acids in the GIT [45]. Although no effect of PF on pH in the gu<sup>t</sup> was found in our study, we found the beneficial effect of PF feeding on apparent total tract digestibility of DM, Zn, and Cu, but ATTD of Fe and Mn was considerably decreased. Similarly, fibre source differently affected soluble content of TEs in digesta of the ileum and/or jejunum. Several ligands of different types of dietary fibres are responsible for the metal binding and the binding strength to different metal ions [41]. We can only speculate that the physicochemical properties of PF might differently affect the solubility of TEs in the GIT and their digestibility. Potato starch from potato fibre has a higher viscosity and longer texture in comparison to corn starch [46] used in the C and ZnGly diets, which could influence conditions for mineral absorption in the GIT. Different effects of digestible starch on the apparent intestinal absorption of Fe and Zn were also observed in pigs [47]. Further investigation is needed to elucidate the mechanism of action of potato fibre in mineral absorption and the different effect of PF on the digestibility of TEs.

The amount of trace minerals available for absorption (bioaccessible or soluble amount) depends on the concentration of TEs in the diet, feed composition, and the presence of ligand enhancers or inhibitors of mineral absorption in the GIT [20]. Conditions increasing the solubility of trace minerals or protecting them from interaction with inhibitors in the intestinal lumen are generally beneficial for their absorption and uptake by the apical surface of enterocytes in the small intestine [37]. On the other hand, soluble inorganic species of TEs are more sensible to form insoluble chelate compounds with various chelators at the pH of the gu<sup>t</sup> lumen [48]. In our study, the reduced ATTD of Zn in the ZnGly treatment might be a result of decreased soluble Zn in the ileal digesta of pigs supplemented with ZnGly. Similarly, the highest soluble Zn content observed in the ileum of piglets fed the PF diet could lead to increased ATTD of Zn.

On the other hand, diet supplementation with PF increased the ATTD of Cu in our piglets, while in vitro solubility of Cu in the simulated gastric phase as well as in situ solubility of Cu in the jejunum decreased. Cu is primarily absorbed through the stomach and small intestine of monogastric animals and gastric acidity promotes the presence of freely-soluble Cu ions [49]. Cu solubility in the gastric phase could predict mineral availability in the rest of the gastrointestinal tract, so decreased solubility of Cu in the simulated in vitro gastric phase could be related to the reduced soluble Cu percentage in the jejunum (in situ). However, we found interactions between both dietary sources which

might influence the ATTD of Cu in piglets. It has been reported that in vitro solubility of Cu might not accurately represent the in vivo bioavailability of Cu due to either disassociation or chemical shifts in Cu along the intestinal tract of broilers [50]. In any case, it should be stressed that Zn source had no effects on either Cu solubility or Cu digestibility in the GIT of our piglets, which is an important fact concerning the competitive antagonism for absorption between those two trace minerals.

Feed supplementation with PF negatively influenced the total apparent digestibility of Fe and decreased Fe absorption from the ileum of our piglets. On the other hand, in situ solubility of Fe increased in the ileum of piglets fed the PF diet. Solubility of Fe could be affected by changes in intestinal pH in the presence of PF in the diet, but no effect of PF on pH was observed in our study. It seems that even though in situ solubility of Fe increased in the ileum, the PF treatment interactions with other feed components along the digestive tract decreased the total digestibility of Fe. Moreover, it has been identified several complexes of Fe which are soluble, but unabsorbable in the gut, using an in vitro method measuring Fe solubility to predict Fe bioavailability [51]. In vitro and in situ solubility of Fe could be affected by Zn source, because ZnGly supplementation decreased Fe solubility in both trials. Similarly, in vitro and in situ solubility of Mn was affected by both dietary sources, but the effect is inconsistent. It seems that interaction between Zn and fibre sources led to the reduced apparent digestibility of Mn. Therefore, further investigation is needed to fully understand the effects of ZnGly and potato fibre on Fe and Mn absorption and interactions, and also competitive inhibition between the minerals in the GIT.

Another important factor affecting the solubility of mineral complexes in the GIT is the pH level of its various parts. Complexes formed by phytic acid and TEs were soluble under the acidic conditions in the in vitro simulated gastric phase, and therefore the highest in vitro soluble content of each trace mineral were recorded in this phase. In contrast, the solubility of phytate complexes with Fe increases above pH 4 and they are more insoluble at gastric pH [5], resulting in reduced in vitro soluble content of Fe in GP in our study. Moreover, ZnGly in the diet even reduced Fe in vitro solubility in GP, but increased soluble Mn. It seems that PF in the diet could affect mineral solubility due to increased pH in simulated in vitro GP, but our study design meant that we did not measure pH in the stomachs of our piglets to confirm this assumption in vivo. On the other hand, increased Mn digestibility in the duodenum after ZnGly supplementation could probably be a result of higher in vitro solubility of Mn in the GP, because mineral solubility in the gastric phase then dictates the availability of those minerals in the rest of the gastrointestinal tract. In situ solubility of Mn in the small intestine segments were affected by both dietary sources.

In vitro solubility is one of the methods of assessing the bioaccessible amounts of TEs based on the determination of soluble TEs under simulated physiological conditions [1]. Although bioaccessibility of TEs evaluated by in vitro methods is an important step in the assessment of trace mineral bioavailability, more important is the amount of TEs which is absorbed into the systemic circulation, and converted to the physiologically active species [2]. In vitro methods are used to identify possible physicochemical properties of feed compounds that could contribute to explain differences in mineral absorption [51]. The prediction of bioavailability of TEs by means of in vitro digestion assay is only relative because it does not exactly mimic the in vivo digestion due to physiological and environmental factors [52], but the solubility of TEs determined in in vitro experiments might represent the bioaccessible amount of TEs in the dietary treatments for pigs. In vitro simulated digestion in the SI phase significantly decreased concentrations of soluble TEs except Cu, which could have resulted in no significant differences between the treatments in this phase. At the intestinal pH value used in simulated in vitro assay (pH 6.8) or during passage through the small intestine, mineral complexes becoming insoluble and precipitated, thus decreasing mineral absorption due to de novo complexation [5,6].

The change in pH and introduction of more enzymes in the simulated small intestinal phase activated a series of reactions leading to complexation, adsorption, and precipitation of TEs, and decreased bioaccessibility of Zn, Mn, and Fe [53]. Phytic acid in the

cereal-based diet chelated Zn, Fe, and Mn (but not Cu), forming insoluble and undigestible complexes [54]. Even though phytic acid binds Cu more strongly that Zn in vitro, complexes with Cu are soluble over a wide pH range and are less precipitated at neutral pH [55]. Moreover, no inhibitory effect of phytic acid on Cu absorption in vivo has been confirmed [56,57] nor on good solubility of Cu-phytate complexes in the intestinal lumen [58,59]. Enzyme phytases originating from plants, microorganisms, and animal tissues catalyse phytate hydrolysis to inorganic phosphate [60]. Endogenous phytases can degrade phytates, and their complexes with TEs in pig nutrition are phytases generated by the small intestinal mucosa, microbial phytase presents mainly in the large intestine, and intrinsic plant phytase derived from certain feedstuffs [61]. We assume that during our in vitro simulated digestion, hydrolysis of phytates and their complexes could take place only through intrinsic plant phytase activity, and therefore the lower in vitro soluble concentrations of TEs in the SI phase was a result of the strong proteolytic activity of proteases in the pancreatin solution at intestinal pH, which broke down the intrinsic plant feed phytases to result in increased formation of insoluble mineral-phytate complexes and their precipitation [62,63]. On the other hand, gut-derived microfloral and mucosal phytases, which are more stable at higher pH in the duodenum than intrinsic plant feed phytases, could have hydrolysed mineral-phytate complexes in the small intestine of our piglets in vivo, so the in situ solubility of TEs and also the apparent digestibility of TEs did not significantly affect the small intestinal segments of the piglets.

Our inconsistent results as well as outcomes of other studies [64] indicated that in vitro mineral solubility to predict bioavailability of TEs from their different sources may sometimes be unsatisfactory and misleading. Discrepancy and poor correlation between soluble content and total apparent digestibility of TEs could be caused by different total and soluble content of TEs in intestinal digesta collected only one-time at the end of the study (in situ soluble TEs) in comparison to the mineral concentration in pooled faeces which were collected continually 5 days (in vivo ATTD). However, it has been concluded that the variability of apparent nutrient digestibility derived after slaughtering was similar to that found with cannulated animals [65]. Moreover, except for dietary factors affecting the mineral bioavailability in vitro and in vivo, there are various physiological factors influencing the trace mineral digestibility in animals. Since the physiological factors including animal species, sex, age, state of growth or physiological and nutritional status of animals are not present in in vitro systems, they cannot affect in vitro digestibility [2].

Various in vitro solubility techniques used for estimating mineral bioaccessibility indicated either a good or poor correlation between in vitro solubility and in vivo bioavailability of Zn and Fe, depending on used in vitro conditions and chemical species of dietary TEs [3,52,65]. As the good indicator of Zn bioavailability from different Zn sources seems to be their solubility in pH 5 and 2 buffers for poultry; however, an inverse relationship was found [14]. On the other hand, the positive relationship was found between Cu bioavailability and Cu solubility in pH 2 buffer [66]. Therefore, in vitro studies for measuring bioaccessibility and/or bioavailability of TEs are not suitable for fully substituted in vivo studies, and should be regarded as screening methods to help us identify dietary factors affecting the absorption of TEs and to study their interactions. There is a need for more validation studies in which the in vivo results are compared to in vitro results.
