**4. Discussion**

Twelve-week feed supplementation with 80 mg of Zn/kg did not affect plasma Zn concentrations measured once a month compared to control. However, after the 120 day feeding period with differing Zn dietary sources, the plasma Zn concentration increased in lambs fed the diet supplemented with ZnProt. In many studies, long-term supplementation of the basal diet with a low Zn concentration range (10–30 mg of Zn/kg) as well as Zn dietary source affected neither plasma nor tissue Zn concentrations in ruminants [6,12,31–33]. We decided to supplement the TMR with close to the proposed total maximum contents of Zn for ruminants in the EU [34] to contain a total of 80 mg Zn/kg of complete diet from different Zn sources. Zn plasma levels reflected dietary Zn concentrations, when sheep were fed with Zn-marginal or -deficient diets containing only 14–22 mg Zn/kg DM [17,35,36]. Our results indicate that the unsupplemented TMR containing 30 mg of Zn/kg was sufficient to meet the physiological requirements for growing lambs. Because the offered TMR was not Zn-deficient and plasma Zn concentrations were maintained within a relatively narrow value span because of efficient homeostatic regulation [37], differences in plasma Zn levels between the dietary treatments were not significant. Although we found a time effect on Zn plasma concentrations, a long period of Zn supplementation caused changes in Zn absorption and excretion to maintain homeostasis; therefore, no differences in Zn bioavailability were detected.

Although Zn plasma seems to not be a reliable marker for assessment of Zn bioavailability [3,12], and we found positive correlation between Zn concentrations in plasma and pancreas, liver, and kidney. Zn concentrations in liver, kidney, pancreas, and bone are considered as suitable markers for assessment of Zn bioavailability in ruminants [11,31,38]. However, increased Zn concentrations were observed mainly in these tissues in ruminants fed diets supplemented with high Zn concentrations (300–1400 mg Zn/kg), with the greatest Zn deposition in plasma and/or tissues in animals fed diets enriched with organic Zn sources [33,39,40]. Differences between Zn feed additives have not been found in tissue mineral deposition using low or normal Zn supplementation levels [6,31–33,41]. Despite the fact that Zn concentrations in tissues are controlled by homeostatic changes in Zn absorption and/or endogenous excretion [4,37], and we supplemented our lambs' diet with up to 80 mg of Zn/kg, increased Zn concentrations were found in the kidneys of lambs fed the ZnProt diet compared to other supplemented or unsupplemented lambs, and Zn levels were elevated in the liver of lambs receiving ZnSO4 treatment compared to controls but with no differences between Zn sources. Calves receiving ZnSO4 at 20 mg of Zn/kg for 56 or 98 days had higher Zn content in their liver than animals fed ZnProt, but after increasing supplemental Zn levels to 500 mg Zn/kg, greater absorption and retention of Zn from ZnProt were observed, resulting in increased Zn deposition in liver, kidney, and plasma [31]. Our results sugges<sup>t</sup> that feed supplementation with Zn from different sources at 80 mg of Zn/kg of diet resulted in Zn being absorbed and utilized at similar rates, and higher Zn levels in the kidneys of lambs fed the ZnProt diet may indicate some differences in the post-absorptive metabolism of Zn from an organic source [9,16,31].

Zinc as an activator of the enzyme SOD as well as other antioxidant proteins and molecules contributes to the proper functioning of the antioxidant defense system [5,42,43]. Regardless of Zn source, Zn supplementation improves the antioxidant status of ruminants due to the increased antioxidant activity of SOD and glutathione peroxidase (GPx) [7,14,44], while in our study, the total antioxidant status improved in the pancreas and the SOD activity in the liver of all supplemented lambs. Intake of supplemented diets with zinc sulphate and Zn glycinate significantly increased liver activity of total SOD, and the highest Cu/Zn SOD activity was determined in the liver of lambs given the ZnGly diet only. It seems that ZnGly could increase the antioxidant activity of Cu/Zn SOD in the liver, which is supported by the hypothesis of higher Zn availability from ZnGly maintaining the normal enzyme activity in the body; however, ZnGly treatment affected Cu/Zn SOD and/or ALP activity in the liver and serum of monogastric animals [45–47]. Zn supplementation from organic Zn sources increased Cu/Zn SOD activity as well as Zn concentrations in the liver

of ruminants compared to ZnSO4 [31,33,48,49]. Surprisingly, we found remarkably reduced activity in both antioxidant enzymes, SOD and GPx, in the liver and/or blood as well as plasma metallothionein concentration in lambs supplemented with ZnProt; however, the lambs receiving ZnProt treatment had the highest Zn concentration in kidney and plasma after the 120 d feeding period. This could indicate that Zn from ZnProt was not associated with SOD enzyme synthesis and may be metabolized differently from other Zn sources [31].

The most appropriate criteria for estimating the relative bioavailability (RVB) of zinc in mature ruminants is Zn concentration in the liver, kidney, and pancreas or liver metallothionein concentration [3,31,39,50], when the RVB of zinc sources was expressed relative to zinc sulphate as a standard source. We decided to use Zn concentrations in liver, kidney, and pancreas tissue to estimate the RVB of Zn sources; however, the tissue Zn deposition in particular did not differ significantly from the other supplemented treatments. Since the bioavailability of minerals is defined as the proportion of ingested element from feed which is utilized for specific physiological and biochemical functions at the site of action [1,51], we also used the liver activity of Cu/Zn SOD to estimate RVB, as that was where the significant differences between the individual treatments were found. Our results show that the RBV of Zn did not differ among the Zn sources, because there were no significant differences between their linear regression slopes. The best parameter for bioavailability assessment appears to be the activity of Cu/Zn SOD in the liver, as the highest coefficient of determination for this parameter was found there. This is in accord with other studies reporting no differences in Zn utilization and RVB among Zn sources in ruminants [33,39,41,52]. Potentially better bioavailability of organic Zn chelates or complexes in ruminants has been reported in a few studies based on improved gu<sup>t</sup> absorption, tissue retention of Zn, and/or higher activity of Cu/Zn SOD in liver [17,48]. Cao et al. [39] reported that Zn proteinate was a more available source of Zn for lambs than ZnSO4 and other organic Zn sources due to the higher Zn uptake by kidney and pancreas tissues. Although we also recorded higher Zn concentrations in kidney and plasma of lambs fed the ZnProt diet and liver activity of SOD in ZnGly treatment, we assumed similar relative bioavailability of all Zn sources used based on our linear regression slope ratio comparisons.

Potentially greater absorption of Zn from organic sources, slower release of Zn from Zn chelates or complexes and differing post-absorptive metabolism may affect mineral tissue deposition and mineral status in ruminants. Zn, Cu, Mn, and Fe are chemically similar, so interaction of these elements appears to be tissue-specific due to the competition among the minerals sharing the same transporter systems at the level of membrane transport [53,54]. It has been suggested that Zn supplementation stimulates the production of metallothioneins (MTs) in the intestine and other tissues [55]. In our study Zn supplementation affected mineral status in lambs due to the different mineral tissue deposition. Intake of the diets supplemented with Zn from both organic sources resulted in increased Cu deposition in the pancreas and reduced Mn uptake by the heart muscle. ZnProt supplementation reduced plasma and hepatic Cu concentrations, which could indicate sequestration of dietary Cu in intestinal MT induced by feeding with the ZnProt diet. Low intracellular available Cu bound to MT could affect Fe and Mn transport through the Cu-dependent protein hephaestin and a common transporter, ferroportin [56,57]. Unfortunately, we did not measure MT concentrations in the intestine mucosa, so our results cannot clearly indicate binding of Cu to MT. Further investigation is needed to elucidate whether Zn intake from ZnProt can induce a high metallothionein level in the intestinal mucosa and post-absorptive metabolism of ZnProt as well.
