This study was conducted to evaluate the impact of short-term dietary supplementation with different zinc nanoparticles (Zn NPs) on plasma mineral status, antioxidant response, hematological parameters, apparent nutrient digestibility, intestinal microbial population and bacterial enzymatic activity in growing lambs. Twenty-seven male lambs (Improved Valachian, initial weight 21.2 ± 1.1 kg) aged 5 months were randomly assigned to one of three treatments (n = 9) for 28 days. Each group was fed the identical basal diet with either no supplemental Zn (control group, CON) or supplemented with commercial ZnO nanoparticles (ZnO NPs) or synthesized zinc phosphate nanoparticles (ZnP NPs) at the same dose of 80 mg Zn/kg diet. The results showed that the dietary treatment had no significant effects on the hematological and selected biochemical parameters, plasma metalloprotein level and apparent nutrient digestibility. On day 14, intake of ZnP NPs significantly elevated Zn (p < 0.01) and Fe concentration (p < 0.05) in plasma compared to the CON and ZnO NPs groups. Regardless of the source, supplementation with Zn NPs increased plasma total antioxidant status on day 28 compared to the CON group (p < 0.01), but it did not affect the lipid peroxidation in plasma and activity of antioxidant enzymes in blood. The intake of Zn NPs significantly influenced fecal microbial communities; specifically, reduced populations within the Ruminococcus-Eubacterium-Clostridium cluster and/or the Bacteroides/Prevotella group were observed compared to the CON group, especially at the end of the experiment (p < 0.001). Furthermore, the activity of bacterial enzymes, such as β-glucuronidase, N-acetyl-glucosaminidase and β-galactosidase, was significantly decreased during the experiment in both groups receiving Zn NPs. In conclusion, short-term feeding of diets supplemented with different Zn NPs at 80 mg Zn/kg diet improved total antioxidant status in plasma and did not induce oxidative stress in growing lambs. Dietary Zn NPs were also found to be very effective in altering gut microbiota composition and inhibiting bacterial enzyme activity.

Department of Inorganic Chemistry Faculty of Science Palacky University Olomouc Czechia

Institute of Animal Physiology Centre of Biosciences of the Slovak Academy of Sciences Košice Slovakia

University of Veterinary Medicine and Pharmacy in Košice Košice Slovakia

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Grešáková Ľ, Tokarčíková K, Čobanová K. Bioavailability of dietary zinc sources and their effect on mineral and antioxidant status in lambs. Agriculture. (2021) 11:1093. doi: 10.3390/agriculture11111093 DOI

Wo Y, Jin Y, Gao D, Ma F, Ma Z, Liu Z, et al. Supplementation with zinc proteinate increases the growth performance by reducing the incidence of diarrhea and improving the immune function of dairy calves during the first month of life. Front Vet Sci. (2022) 9:911330. doi: 10.3389/fvets.2022.911330, PubMed DOI PMC

Duffy R, Yin M, Redding LE. A review of the impact of dietary zinc on livestock health. J Trace Elem Miner. (2023) 5:100085. doi: 10.1016/j.jtemin.2023.100085 DOI

Liu J, Yu X, Ma F, Wo Y, Jin Y, Hashem NM, et al. Early supplementation with zinc proteinate does not change rectal microbiota but increases growth performance by improving antioxidant capacity and plasma zinc concentration in preweaned dairy calves. Front Vet Sci. (2023) 10:1236635. doi: 10.3389/fvets.2023.1236635, PubMed DOI PMC

Tokarczyk J, Koch W. Dietary Zn – recent advances in studies on its bioaccessibility and bioavailability. Molecules. (2025) 30:2742. doi: 10.3390/molecules30132742, PubMed DOI PMC

National Research Council (NRC) . Nutrient requirements of small ruminants: Sheep, goats. Cervids, and New World camelids. 1st ed. Washington, DC: The National Academics Press; (2007).

Bhagat S, Singh S. Nanominerals in nutrition: recent developments, present burning issues and future perspectives. Food Res Int. (2022) 160:111703. doi: 10.1016/j.foodres.2022.111703, PubMed DOI

Patra A, Lalhriatpuii M. Progress and Prospect of essential mineral nanoparticles in poultry nutrition and feeding–a review. Biol Trace Elem Res. (2020) 197:233–53. doi: 10.1007/s12011-019-01959-1, PubMed DOI

Zhou H, McClements DJ. Recent advances in the gastrointestinal fate of organic and inorganic nanoparticles in foods. Nano. (2022) 12:1099. doi: 10.3390/nano12071099, PubMed DOI PMC

Michalak I, Dziergowska K, Alagawany M, Farag MR, El-Shali NA, Tuli HS, et al. The effect of metal-containing nanoparticles on the health, performance and production of livestock animals and poultry. Vet Q. (2022) 42:68–94. doi: 10.1080/01652176.2022.2073399 PubMed DOI PMC

Swain PS, Rao SBN, Rajendran D, Dominic G, Selvaraju S. Nano zinc, an alternative to conventional zinc as animal feed supplement: a review. Anim Nutr. (2016) 2:134–41. doi: 10.1016/j.aninu.2016.06.003, PubMed DOI PMC

Alijani K, Rezaei J, Rouzbehan Y. Effect of nano-ZnO, compared to ZnO and Zn-methionine, on performance, nutrient status, rumen fermentation, blood enzymes, ferric reducing antioxidant power and immunoglobulin G in sheep. Anim Feed Sci Technol. (2020) 267:114532. doi: 10.1016/j.anifeedsci.2020.114532 DOI

Mandal AK, Katuwal S, Tettey F, Gupta A, Bhattarai S, Jaisi S, et al. Current research on zinc oxide nanoparticles: synthesis, characterization, and biomedical applications. Nano. (2022) 12:3066. doi: 10.3390/nano12173066, PubMed DOI PMC

Yang J, Xiong D, Long M. Zinc oxide nanoparticles as next-generation feed additives: bridging antimicrobial efficacy, growth promotion, and sustainable strategies in animal nutrition. Nano. (2025) 15:1030. doi: 10.3390/nano15131030, PubMed DOI PMC

Abdelnour SA, Alagawany M, Hashem NM, Farag MR, Alghamdi ES, Hassan FU, et al. Nanominerals: fabrication methods, benefits and hazards, and their applications in ruminants with special reference to selenium and zinc nanoparticles. Animals. (2021) 11:1916. doi: 10.3390/ani11071916, PubMed DOI PMC

Yusof HM, Mohamad R, Zaidan UH, Rahman NAA. Microbial synthesis of zinc oxide nanoparticles and their potential application as an antimicrobial agent and a feed supplement in animal industry: a review. J Anim Sci Biotechnol. (2019) 10:57. doi: 10.1186/s40104-019-0368-z, PubMed DOI PMC

Baholet D, Skalickova S, Batik A, Malyugina S, Skladanka J, Horký P. Importance of zinc nanoparticles for the intestinal microbiome of weaned piglets. Front Vet Sci. (2022) 9:852085. doi: 10.3389/fvets.2022.852085, PubMed DOI PMC

Riazi H, Rezaei J, Rouzbehan Y. Effects of supplementary nano-ZnO on in vitro ruminal fermentation, antioxidant, and microbial biomass. Turk J Vet Anim Sci. (2019) 43:737–46. doi: 10.3906/vet-1905-48 DOI

Mohammed AM, Mohammed M, Oleiwi JK, Ihmedee FH, Adam T, Betar BO, et al. The anticancer, antioxidant, and antimicrobial properties of zinc oxide nanoparticles: a comprehensive review. Nano TransMed. (2025) 4:100097. doi: 10.1016/j.ntm.2025.100097 DOI

Singh S. Zinc oxide nanoparticles impacts: cytotoxicity, genotoxicity, developmental toxicity, and neurotoxicity. Toxicol Mech Methods. (2019) 29:300–11. doi: 10.1080/15376516.2018.1553221, PubMed DOI

Kumari S, Raturi S, Kulshrestha S, Chauhan K, Dhingra S, András K, et al. A comprehensive review on various techniques used for synthesizing nanoparticles. J Mater Res Technol. (2023) 27:1739–63. doi: 10.1016/j.jmrt.2023.09.291 DOI

Horký P, Skalickova S, Urbanková L, Baholet D, Kociova S, Bytesnikova Z, et al. Zinc phosphate-based nanoparticles as a novel antibacterial agent: in vivo study on rats after dietary exposure. J Anim Sci Biotechnol. (2019) 10:17. doi: 10.1186/s40104-019-0319-8, PubMed DOI PMC

Kociova S, Dolezelikova K, Horky P, Skalickova S, Baholet D, Bozdechova L, et al. Zinc phosphate-based nanoparticles as alternatives to zinc oxide in diet of weaned piglets. J Anim Sci Biotechnol. (2020) 11:59. doi: 10.1186/s40104-020-00458-x, PubMed DOI PMC

Petrič D, Mikulová K, Bombárová A, Batťanyi D, Čobanová K, Kopel P, et al. Efficacy of zinc nanoparticle supplementation on ruminal environment in lambs. BMC Vet Res. (2024) 20:425. doi: 10.1186/s12917-024-04281-8, PubMed DOI PMC

Holzwarth U, Gibson N. The Scherrer equation versus the 'Debye-Scherrer equation'. Nat Nanotechnol. (2011) 6:534. doi: 10.1038/nnano.2011.145, PubMed DOI

The European Commision (EC) . Commision implementing regulation (EU) 2016/1095. Off J Eur Union. (2016), 182:7–27.

Jo C, Ahn DU. Fluorometric analysis of 2-thiobarbituric acid reactive substances in Turkey. Poult Sci. (1998) 77:475–80. doi: 10.1093/ps/77.3.475, PubMed DOI

AOAC International . Official methods of analysis. 17th ed. Gaithersburg, MD: AOAC International; (2000).

Gresakova L, Venglovska K, Cobanova K. Dietary manganese source does not affect Mn, Zn and cu tissue deposition and the activity of manganese-containing enzymes in lambs. J Trace Elem Med Biol. (2016) 38:138–43. doi: 10.1016/j.jtemb.2016.05.003, PubMed DOI

Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. (1991) 74:3583–97. doi: 10.3168/jds.S0022-0302(91)78551-2, PubMed DOI

Van Keulen J, Young BA. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J Anim Sci. (1977) 44:282–7. doi: 10.2527/jas1977.442282x DOI

Bergero D, Préfontaine C, Miraglia N, Peiretti PG. A comparison between the 2N and 4N HCL acid-insoluble ash methods for digestibility trials in horses. Animal. (2009) 3:1728–32. doi: 10.1017/S1751731109990656, PubMed DOI

Franks AH, Harmsen HJ, Raangs GC, Jansen GJ, Schut F, Welling GW. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl Environ Microbiol. (1998) 64:3336–45. doi: 10.1128/aem.64.9.3336-3345.1998, PubMed DOI PMC

Manz W, Amann R, Ludwig M, Vancanneyt M, Schleiffer KH. Application of a suite of 16s rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment. Microbiology. (1996) 142:1097–106. doi: 10.1099/13500872-142-5-1097, PubMed DOI

Labuda J, Barek J, Gajdosechova Z, Goenaga-Infante H, Johnson LJ, Mester Z, et al. Analytical chemistry of engineered nanomaterials: part 1. Scope, regulation, legislation, and metrology (IUPAC technical report). Pure Appl Chem. (2023) 95:133–63. doi: 10.1515/pac-2021-1001, DOI

Youn SM, Choi SJ. Food additive zinc oxide nanoparticles: dissolution, interaction, fate, cytotoxicity, and oral toxicity. Int J Mol Sci. (2022) 23:6074. doi: 10.3390/ijms23116074, PubMed DOI PMC

Killilea DW, Siekmann JH. The role of zinc in the etiology of Anemia In: Karakochuk CD, Zimmermann MB, Moretti D, Kraemer K, editors. Nutritional Anemia. Switzerland: Springer Nature; (2022). 187–94.

Pal RP, Mani V, Mir SH, Sharma A, Sarkar S. Replacing inorganic source of zinc with zinc hydroxy chloride: effects on health status, hemato-biochemical attributes, antioxidant status, and immune responses in pre-ruminant crossbred calves. Biol Trace Elem Res. (2025) 203:2001–12. doi: 10.1007/s12011-024-04317-y PubMed DOI

Hosseini-Vardanjani SF, Rezaei J, Karimi-Dehkordi S, Rouzbehan Y. Effect of feeding nano-ZnO on performance, rumen fermentation, leukocytes, antioxidant capacity, blood serum enzymes and minerals of ewes. Small Rumin Res. (2020) 191:106170. doi: 10.1016/j.smallrumres.2020.106170 DOI

Kondaiah P, Yaduvanshi PS, Sharp PA, Pullakhandam R. Iron and zinc homeostasis and interactions: does enteric zinc excretion cross-talk with intestinal Iron absorption? Nutrients. (2019) 11:1885. doi: 10.3390/nu11081885, PubMed DOI PMC

Goff JP. Invited review: mineral absorption mechanisms, mineral interactions that affect acid-base and antioxidant status, and diet considerations to improve mineral status. J Dairy Sci. (2018) 101:2763–813. doi: 10.3168/jds.2017-13112, PubMed DOI

Oliveira DC, Nogueira-Pedro A, Santos EW, Hastreiter A, Silva GB, Borelli P, et al. A review of select minerals influencing the haematopoietic process. Nutr Res Rev. (2018) 31:267–80. doi: 10.1017/S0954422418000112, PubMed DOI

Hara T, Takeda T, Takagishi T, Fukue K, Kambe T, Fukada T. Physiological roles of zinc transporters: molecular and genetic importance in zinc homeostasis. J Physiol Sci. (2017) 67:283–301. doi: 10.1007/s12576-017-0521-4, PubMed DOI PMC

Stewart AJ, Blindauer CA, Berezenko S, Sleep D, Sadler PJ. Interdomain zinc site on human albumin. Proc Natl Acad Sci. (2003) 100:3701–6. doi: 10.1073/pnas.0436576100, PubMed DOI PMC

Vigh A, Criste AD, Corcionivoschi N, Gerard C. Rumen solubility of copper, manganese and zinc and the potential link between the source and rumen function: a systematic review. Agriculture. (2023) 13:2198. doi: 10.3390/agriculture13122198 DOI

Marreiro DN, Cruz KJC, Morais JBS, Beserra JB, Severo JS, Oliveira ARS. Zinc and oxidative stress: current mechanisms. Antioxidants. (2017) 6:24. doi: 10.3390/antiox6020024 PubMed DOI PMC

Lin Y, Wang Y, Li P. Mutual regulation of lactate dehydrogenase and redox robustness. Front Physiol. (2022) 13:1038421. doi: 10.3389/fphys.2022.1038421, PubMed DOI PMC

Najafzadeh H, Ghoreishi SM, Mohammadian B, Rahimi E, Afzalzadeh MR, Kazemivarnamkhasti M, et al. Serum biochemical and histopathological changes in liver and kidney in lambs after zinc oxide nanoparticles administration. Vet World. (2013) 6:534–7. doi: 10.5455/vetworld.2013.534-537 DOI

Lim JS, Yang JH, Chun BY, Kam S, Jacobs DR, Lee DH. Is serum gamma-glutamyltransferase inversely associated with serum antioxidants as a marker of oxidative stress? Free Radic Biol Med. (2004) 37:1018–23. doi: 10.1016/j.freeradbiomed.2004.06.032, PubMed DOI

Mohamed AH, Mohamed MY, Ibrahim K, Abd-El-Ghany FTF, Mahgoup AAS. Impact of nano-zinc oxide supplementation on productive performance and some biochemical parameters of ewes and offspring. Egypt J Sheep Goat Sci. (2017) 12:49–64. doi: 10.21608/ejsgs.2017.26308 DOI

Walker RB, Everette JD. Comparative reaction rates of various antioxidants with ABTS radical cation. J Agric Food Chem. (2009) 57:1156–61. doi: 10.1021/jf8026765, PubMed DOI

Tapiero H, Tew KD. Trace elements in human physiology and pathology: zinc and metallothioneins. Biomed Pharmacother. (2003) 57:399–411. doi: 10.1016/S0753-3322(03)00081-7, PubMed DOI

Hatfield PG, Robinson BL, Minikhiem DL, Kott RW, Roth NI, Daniels JT, et al. Serum α-tocopherol and immune function in yearling ewes supplemented with zinc and vitamin E. J Anim Sci. (2002) 80:1329–34. doi: 10.2527/2002.8051329x PubMed DOI

Anam MS, Astuti A, Widyobroto BP, Agus A. Effect of dietary supplementation with zinc-methionine on ruminal enzyme acivities, fermentation characteristics, methane production, and nutrient digestibility: An PubMed DOI PMC

Garg AK, Mudgal V, Dass RS. Effect of organic zinc supplementation on growth, nutrient utilization and mineral profile in lambs. Anim Feed Sci Technol. (2008) 144:82–96. doi: 10.1016/j.anifeedsci.2007.10.003 DOI

Van Valin KR, Genther-Schroeder ON, Carmichael RN, Blank CP, Deters EL, Hartman SJ, et al. Influence of dietary concentration and supplementation zinc source on nutrient digestibility, zinc absorption, and retention in sheep. J Anim Sci. (2018) 96:5336–44. doi: 10.1093/jas/sky384 PubMed DOI PMC

Esfiokhi SHM, Norouzian MA, Najafi A. The effect of different zinc sources on biochemical parameters, intestinal morphology, carcass characteristics and performance in finishing lambs. Biol Trace Elem Res. (2024) 202:175–81. doi: 10.1007/s12011-023-03675-3, PubMed DOI

Bujňáková D, Kucková K, Váradyová Z, Plachá I, Strompfová V, Bohm J, et al. Effects of dietary zinc and/or an herbal mixture on intestinal microbiota and barrier integrity in lambs. Agriculture. (2023) 13:1819. doi: 10.3390/agriculture13091819 DOI

Zhang Y, Choi SH, Nogoy KM, Liang S. Review: the development of the gastrointestinal tract microbiota and intervention in neonatal ruminants. Animals. (2021) 15:100316. doi: 10.1016/j.animal.2021.100316, PubMed DOI

Chang MN, Wei JY, Hao LY, Ma FT, Li HY, Zhao SG, et al. Effects of different types of zinc supplement on the growth, incidence of diarrhea, immune function, and rectal microbiota of newborn dairy calves. J Dairy Sci. (2020) 103:6100–13. doi: 10.3168/jds.2019-17610, PubMed DOI

Faulkner MJ, Wenner BA, Solden LM, Weiss WP. Source of supplemental dietary copper, zinc, and manganese affects fecal microbial relative abundance in lactating dairy cows. J Dairy Sci. (2017) 100:1037–44. doi: 10.3168/jds.2016-11680, PubMed DOI

Chen C-W, Hsu C-Y, Lai S-M, Syu W-J, Wang T-Y, Lai P-S. Metal nanobullets for multidrug resistant bacteria and biofilms. Adv Drug Deliv Rev. (2014) 78:88–104. doi: 10.1016/j.addr.2014.08.004, PubMed DOI

Siddiqi KS, Ur Rahman A, Tajuddin, Husen A. Properties of zinc oxide nanoparticles and their activity against microbes. Nanoscale Res Lett. (2018) 13:141. doi: 10.1186/s11671-018-2532-3 PubMed DOI PMC

Oh H-J, Park Y-J, Cho JH, Song M-H, Gu B-H, Yun W, et al. Changes in diarrhea score, nutrient digestibility, zinc utilization, intestinal immune profiles, and fecal microbiome in weaned piglets by different forms of zinc. Animals. (2021) 11:1356. doi: 10.3390/ani11051356, PubMed DOI PMC

Gao S, Sun R, Singh R, So SY, Chan CTY, Savidge T, et al. The role of gut microbial β-glucuronidase in drug disposition and development. Drug Discov Today. (2022) 27:103316. doi: 10.1016/j.drudis.2022.07.001, PubMed DOI PMC

Glover JS, Ticer TD, Engevik MA. Characterizing the mucin-degrading capacity of the human gut microbiota. Sci Rep. (2022) 12:8456. doi: 10.1038/s41598-022-11819 PubMed DOI PMC

Dubinsky V, Reshef L, Rabinowitz K, Yadgar K, Godny L, Zonensain K, et al. Dysbiosis in metabolic genes of the gut microbiomes of patients with an ileo-anal pouch resembles that observed in Crohn's disease. mSystems. (2021) 6:e00984–20. doi: 10.1128/mSystems.00984-20, PubMed DOI PMC

Schomburg D, Salzmann M. N-Acetyl-beta-glucosaminidase In: Schomburg D, Salzmann M, editors. Enzyme Handbook 4. Berlin/Heidelberg, Germany: Springer Nature; (1991). 195–201.

Lallès J-P. Intestinal alkaline phosphatase: novel functions and protective effects. Nutr Rev. (2014) 72:82–94. doi: 10.1111/nure.12082, PubMed DOI