**1. Introduction**

Definitions for mineral bioavailability vary because of the complexity of the issue. From a nutritional point of view, the bioavailability of minerals can be defined as the proportion of ingested trace elements utilized for their specific physiological and biochemical functions at the site of action [1]. Mineral bioavailability includes gastrointestinal digestion, absorption, metabolism, tissue distribution, and bioactivity, but it depends on many factors such as feed composition, co-ingested compounds, mineral dosage and chemical form, and the mineral status of animals [2]. Moreover, due to the substantial impacts of homeostatic mechanisms on the plasma and tissue concentrations of minerals, it is difficult to correctly estimate mineral bioavailability based on the above definition.

Zinc as an essential integral part of many important enzyme systems is involved in keeping the health and performance of ruminants at their maximum [3]. Dietary zinc deficiency could lead to impaired growth, reproduction, and immune dysfunction with increased susceptibility to infections in growing animals [3–5]. Although Zn requirements for growing lambs and calves are recommended at 33 mg Zn/kg of dry matter (DM) in a complete diet, young ruminants require additional Zn to support accretion of body protein during periods of rapid growth, and improvement in growth performance, health and reproduction, mineral and antioxidant status, and immunity in ruminants has been reported after Zn supplementation [3,6–9]. In addition, Zn supplementation with Zn nanoparticles can offer an effective approach to maintaining the high production and health of ruminants due to the improvement in animal reproductive efficiency, immunomodulatory properties, and enhancing the microbial biomass production, while it can also help to reduce methane emission in livestock [10].

**Citation:** Grešáková, L'.; Tokarˇcíková, K.; Cobanová, K. Bioavailability of ˇ Dietary Zinc Sources and Their Effect on Mineral and Antioxidant Status in Lambs. *Agriculture* **2021**, *11*, 1093. https://doi.org/10.3390/ agriculture11111093

Academic Editor: Secundino <sup>L</sup>ópez

Received: 6 October 2021 Accepted: 2 November 2021 Published: 4 November 2021

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The methods of assessment of Zn bioavailability include Zn absorption and retention, plasma or tissue Zn concentrations, animal growth, the biologically active form of Zn, and prevention of signs of Zn deficiency [11]. Intake of Zn from organic sources has been found to increase nutrient digestibility and Zn absorption and retention in lambs compared with inorganic Zn supplementation [7,12,13], but the results of other studies focusing on zinc bioavailability in ruminants are contradictory [14–18]. Interaction of Zn with other metal ions in the diet is a dietary factor that can modify Zn bioavailability. Complex interactions among minerals within the digestive tract can affect the absorption of zinc as well as the feed's effectiveness in promoting the health and productivity of ruminants, but organic Zn chelates might permit Zn and other minerals in the diet to circumvent factors that inhibit absorption of their inorganic forms [17,19]. Therefore, in this study, we decided to estimate the relative bioavailability of different zinc sources used as feed additives in ruminant nutrition based on Zn concentrations as well as the activity of Zn-dependent enzymes in lamb tissues and to investigate the effect of organic Zn sources on the mineral and antioxidant status in lambs.

#### **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 Ethical Committee of the Institute of Animal Physiology of the Slovak Academy of Sciences and by the State Veterinary and Food Office (Ro-4160/13-221).

#### *2.1. Animals, Diets, and Experimental Design*

Thirty-two castrated male lambs (Improved Valachian breed) aged three months were selected from the flock of a commercial farm (Olšavica–Brutovce, Slovakia) with an initial mean body weight 19.7 ± 3.2 kg. The lambs were housed at the Slovak Academy of Sciences Institute of Animal Physiology Research Centre. They were allocated to one of four dietary treatments (*n* = 8) in a completely randomized design and fed an unsupplemented total mix ration (TMR) consisting of grass hay and feed concentrate (600 and 300 g/day, respectively) in the adaptation period lasting four weeks. During the experimental period, lasting 120 days, at four months of age (mean BW 24.7 ± 2.97 kg), the lambs were fed the TMR consisting of grass hay and feed concentrate (800 and 350 g/day, respectively) providing a total of 29.6 mg Zn/kg for the control lambs (C, control). The concentrates for the experimental dietary treatments were supplemented either with zinc sulphate as an inorganic Zn source (ZnSO4; ZnSO4·H2O, Sigma–Aldrich, St. Louis, MO, USA), or organic sources of Zn, zinc chelate of glycine hydrate (ZnGly; Glycinoplex-Zn 26%, Phytobiotics Futterzusatzstoffe GmbH, Eltville, Germany) and zinc chelate of protein hydrolysate (ZnProt; Bioplex®-Zn 15%, Alltech Inc., Nicholasville, KY, USA). Commercially produced Zn organic chelates (ZnGly, ZnProt) are more stable complexes produced by reacting Zn with amino acid hydrate or hydrolyzed protein protecting Zn in the gu<sup>t</sup> from the adverse effects of other feed compounds and minerals and make it more absorbable and available to lambs. The TMRs for all dietary treatments were formulated to meet National Research Council (NRC, 2007) requirements [20], and all supplemented diets (TMRs for the supplemented dietary treatments) were prepared so as to contain a total of 80 mg Zn/kg of a complete diet (as-fed basis). The TMR composition is shown in Table 1.


**Table 1.** Ingredients and chemical composition of the total mix ration (TMR) 1 on a dry matter (DM) basis.

1 Common TMR fed to all lambs during the trial. The control received only the TMR, other dietary treatments received the TMR consisting of grass hay and concentrate supplemented with different Zn sources.

During the adaptation period, the lambs were housed in floor pens with bedding (*n* = 2 lambs/pen, 1.64 × 1.25 m) equipped with an automatic water supplier and plastic feeders. At the beginning of the experimental feeding period, each lamb was housed individually in a pen and fed the TMR consisting of grass hay and concentrate (800 and 350 g/day, respectively). The feed concentrate contained ground barley, wheat bran, and soybean meal as the main ingredients. The addition of the Zn sources to ground concentrate was analytically confirmed (*n* = 8). The analyzed total Zn concentrations in the TMRs of the supplemented experimental treatments were 78.11 mg Zn/kg for ZnSO4, 79.79 mg Zn/kg for the ZnGly, and 77.49 mg Zn/kg for ZnProt treatments. Throughout the experiment, the animals were fed twice a day and had free access to fresh potable water. Once a week each lamb was offered a trace mineral lick without Zn composed of Ca 16.2, Na 316, Mg 32, Cu 0.5, Co 0.06, I 0.02, and Se 0.01 (g/kg). Feed intake from the daily ration was evaluated separately for each lamb, and the body weights were recorded monthly.

#### *2.2. Sample Collection and Analysis*

The total amount of feed refused by each lamb was collected and recorded daily at 8:00 a.m. All refused feed samples were dried and ground to pass through a 1 mm screen and stored for subsequent analysis.

Jugular blood samples were collected into acid-washed heparinized tubes from each lamb once a month during the experimental period. Blood samples were centrifuged at 1180× *g* for 10 min at 4 ◦C, and plasma was removed for mineral analysis. Fresh blood samples were immediately used for the determination of superoxide dismutase activity in erythrocytes. At the end of the experiment, six lambs from each treatment group were slaughtered, and tissue samples were collected. Tissue samples were collected from the left lobe of the liver, the left kidney cortex, apex of the heart and the pancreas as well as from the skeletal muscles *m. longissimus dorsi* and *m. psoas major*. Sampling was conducted from identical locations of each relevant tissue using a ceramic knife. All tissues and plasma samples were stored in acid-washed plastic vials or tubes at −80 ◦C until further analysis.

Dry matter from feed and tissue samples was determined according to the Association of Official Analytical Chemists (AOAC) method [21] by drying samples to a constant weight at 105 ◦C. After drying the samples of diet ingredients at 60 ◦C for 48 h, the samples were analyzed for neutral detergent fiber (NDF) and acid detergent fiber (ADF) using the method proposed by Van Soest et al. [22]. NDF was assayed without heat-stable amylase and expressed inclusive of residual ash. ADF was also expressed inclusive of residual ash. ADF and NDF concentrations were determined using the ANKOM2000 Fibre Analyzer

(Ankom Technology, Macedon, NY, USA). Standard methods [21] were used to analyze ash (No. 942.05), nitrogen (No. 968.06), and crude protein (No. 990.03) using the Kjeldahl method after acid digestion.
