3.1.2. Apparent Digestibility

Feed supplementation with ZnGly decreased the apparent total tract digestibility (ATTD) of Zn (*p* = 0.01), but the apparent digestibility of Zn in the duodenum, jejunum, and ileum did not differ between the dietary treatments (Table 2). Intake of both PF diets increased the total apparent digestibility of Zn (*p* < 0.01) compared to the ZnGly treatments as well as DM digestibility in piglets. The highest value DM digestibility was recorded in the PF + ZnGly treatment compared to the C and ZnGly treatments (Table 5).

Intake of the PF diets increased the total apparent digestibility of Cu (*p* < 0.01), but Cu digestibility did not change in the small intestine. Interaction between Zn and fibre sources affected Cu digestibility in the duodenum (*p* < 0.05), with decreased Cu digestibility in the PF treatment compared to the PF + ZnGly treatment (*p* < 0.05).

On the other hand, PF supplementation significantly reduced Fe digestibility from the ileum as well as decreased the ATTD of Fe (*p* < 0.001), while the apparent digestibility of Fe was not changed in the other small intestine segments.

ZnGly in the diet increased duodenal apparent digestibility of Mn (*p* < 0.05). However, the Zn source affected the apparent duodenal digestibility of Mn, Mn absorption did not differ between the dietary treatments in the jejunum and ileum. The effects of both dietary sources and source interaction were observed on ATTD of Mn, whereby negative values in the parameter were found, with the lowest values in the PF + Gly treatment.

No changes in pH were found in any small intestinal segments in our in vivo experiment (Table 5).


**Table 2.** Apparent digestibility of Zn, Cu, Fe and Mn in the small intestine and total tract of piglets fed diets supplemented with zinc and fibre from different sources.

1 C—cellulose, PF—potato fibre, ZnGly—zinc chelate with glycine. a–b Means within a row with different superscript letters are significantly different (*p* < 0.05) as a result of a Tukey's means comparison (*n* = 6). A,B Means within lines with different superscript letters are significantly different (*p* < 0.05) using by Fisher's LSD post hoc test.

## *3.2. Experiment in situ*

#### In Situ Soluble TEs

Interaction between Zn and fibre sources affected soluble Zn concentration in digesta of each small intestinal segmen<sup>t</sup> (Table 3). Zn solubility in the duodenum and jejunum were not affected by any dietary sources, but the effect of both dietary sources on the soluble concentration of Zn in the ileum was observed. Due to dietary source interaction, significant decreased in situ Zn solubility was observed in the duodenum (*p* < 0.01), but soluble Zn concentration increased in the jejunum (*p* < 0.05) in the PF + Gly treatment compared to the ZnGly and PF treatments. Increased Zn soluble content was observed in the ileal digesta of piglets fed the PF diet in comparison to the other dietary treatments (*p* = 0.001).

Supplementation with PF decreased in situ soluble content of Cu in the jejunum without any significant differences between the treatments.

Zn source affected soluble Fe concentration in the jejunal digesta with significantly decreased soluble content of Fe in the ZnGly treatment to the control treatment (C). Fe solubility in the ileum was affected by both dietary sources (*p* < 0.0001). The highest value was observed in the PF treatment, while the lowest in vitro solubility of Fe was observed in the ZnGly treatment compared to others.

In situ solubility of Mn was affected by both dietary sources in the jejunum and ileum. ZnGly in the diets decreased soluble Mn in the duodenum and ileum, but ZnGly increased Mn solubility in the jejunum. Feed supplementation with PF reduced the soluble content of Mn in the jejunum, on the contrary, increased in vitro solubility of Mn was observed in the ileal digesta in the PF treatment (*p* < 0.01). However, the interaction between both dietary sources affected soluble Mn concentration in the ileal digesta.


**Table 3.** In situ soluble content of Zn, Cu, Fe and Mn from intestinal digesta after dietary treatments with Zn and fibre from different sources.

1 C—cellulose, PF—potato fibre, ZnGly—zinc chelate with glycine. a–c Means within a row with different superscript letters are significantly different (*p* < 0.05) as a result of a Tukey's means comparison (*n* = 6). A–C Means within lines with different superscript letters are significantly different (*p* < 0.05) using by Fisher's LSD post hoc test.

#### *3.3. Experiment In Vitro*

#### 3.3.1. In Vitro Solubility of Zn Sources

The effects of pH on Zn solubility from both Zn sources in different buffers simulating pH in gastric and small intestine digestion were estimated in vitro (Figure 1). The in vitro solubility of Zn from ZnSO4 and ZnGly was affected by pH (*p* < 0.0001) and Zn source (*p* = 0.003), with the lowest Zn solubility at pH 6.8 simulating digestion in the lower part of the small intestine (Tukey's post hoc multiple comparisons test with individual variance computed for pair-wise comparisons in Supplementary materials, Figure S1, Table S1). The solubility of ZnSO4 was found to be significantly higher than Zn solubility from ZnGly (two-way ANOVA with the main effect only, *p* < 0.001, Figure 1).

**Figure 1.** In vitro solubility of Zn from both Zn sources (ZnSO4, ZnGly) subjected to different buffers simulating gastric digestion in 0.2 M Gly-HCl at pH 2.0, or simulating small intestinal digestion in 0.2 M sodium acetate buffer at pH 4.8 and 6.8; Asterisks represent *p-* value classification (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001).

Data were analyzed using two-way ANOVA, followed by the post hoc Tukey's multiple comparisons test, which included the main effect only (pH and Zn source).

#### 3.3.2. In Vitro Simulated Solubility of TEs

In vitro soluble or bioaccessible concentrations of TEs in the experimental diets in each digestion phase are shown in Table 4. Zn, Mn, and Cu were almost totally dissolved in the gastric digestive juice but rapidly decreased solubility of Zn, Mn, and Fe was found in the small intestine phase (SIP). The in vitro solubility of Zn, Fe, and Mn in each experimental diet was observed to be lower in the SIP than in the gastric and large intestinal phases (two-way ANOVA with main effect only, *p* < 0.001); however, Cu solubility did not differ between the simulated digestion phases.

**Table 4.** In vitro solubility of Zn, Cu, Fe, and Mn from the dietary treatments subjected to three-step in vitro simulated digestion assay.


1C—cellulose, PF—potato fibre, ZnGly—zinc chelate with glycine. ab Means within a row with different superscript letters are significantly different (*p* < 0.05) as a result of a Tukey's means comparison (*n* = 9). A–C Means within lines with different superscript letters are significantly different (*p* < 0.05) using Fisher's LSD post hoc test.

The in vitro solubility of Zn did not differ between the dietary treatments in each phase of simulated digestion. Intake of PF diets decreased soluble Cu in the gastric phase (GP) and affected solubility of Fe in the large intestinal phase (LIP).

The Zn source influenced in vitro solubility of Cu in LIP, soluble Fe in GP and LIP, and Mn solubility in gastric digestion phases. Supplementation with organic ZnGly increased in vitro solubility of Cu in LIP (*p* < 0.05) and soluble Mn in GP (*p* < 0.001). Zn source also affected the solubility of Fe in GP and LIP; however, the interaction between Zn and fibre sources was observed in both of these digestion phases. Decreased in vitro solubility of Fe in GIP was observed in the ZnGly treatment, while the highest soluble Fe concentration in LIP was observed in the control treatment compared to other treatments.

#### 3.3.3. In Vitro Dry Matter Digestibility and pH

Although feed supplementation with PF increased in vivo DM digestibility in piglets, in vitro dry matter digestibility (IVDMD) was not influenced by the dietary treatments (Table 5).


**Table 5.** Effects of dietary treatments with added Zn and fibre from different sources on pH and dry matter digestibility subjected to in vivo and in vitro digestion assay.

1 C—cellulose, PF—potato fibre, ZnGly—zinc chelate with glycine. a–c Means within a row with different superscript letters are significantly different (*p* < 0.05) as a result of a Tukey's means comparison, means represent 6 replicates in vivo, 9 replicates in vitro (DM digestibility) or 3 replicates in vitro (pH). A–B Means within lines with different superscript letters are significantly different (*p* < 0.05) using Fisher's LSD post hoc test.

Different effects of Zn and fibre sources on pH values were determined: PF affected pH in the in vitro simulated GP (*p* < 0.01) and Zn source in the SIP (*p* < 0.05). The diets containing PF increased pH in the gastric phase, while ZnGly in the diets slightly decreased pH in the SIP (Table 5). Interaction between both dietary sources affected pH in LIP with increased pH in the Gly and PF treatments.
