{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:30:53Z","timestamp":1787027453581,"version":"build-2736575974"},"reference-count":51,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2024,3,29]],"date-time":"2024-03-29T00:00:00Z","timestamp":1711670400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002848","name":"FONDECYT (Fondo Nacional de Desarrollo Cient\u00edfico y Tecnol\u00f3gico)","doi-asserted-by":"publisher","award":["1210334"],"award-info":[{"award-number":["1210334"]}],"id":[{"id":"10.13039\/501100002848","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Pre-implantation embryos release extracellular vesicles containing different molecules, including DNA. The presence of embryonic DNA in E-EVs released into the culture medium during in vitro embryo production could be useful for genetic diagnosis. However, the vesicles containing DNA might be derived from embryos suffering from apoptosis, i.e., embryos of bad quality. This work intended to confirm that embryos release DNA that is useful for genotyping by evaluating the effect of embryonic apoptosis on DNA content in E-EVs. Bovine embryos were produced by parthenogenesis and in vitro fertilization (IVF). On Day 5, morulae were transferred to individual cultures in an EV-depleted SOF medium. On Day 7, embryos were used to evaluate cellular apoptosis, and each culture medium was collected to evaluate E-EV concentration, characterization, and DNA quantification. While no effect of the origin of the embryo on the apoptotic rate was found, arrested morulae had a higher apoptotic rate. E-EVs containing DNA were identified in all samples, and the concentration of those vesicles was not affected by the origin or quality of the embryos. However, the concentration of DNA was higher in EVs released by the arrested parthenogenetic embryos. There was a correlation between the concentration of E-EVs, the concentration of DNA-positive E-EVs, and the concentration of DNA. There was no negative effect of apoptotic rate on DNA-positive E-EVs and DNA concentration; however, embryos of the best quality with a low apoptotic rate still released EVs containing DNA. This study confirms that the presence of DNA in E-EVs is independent of embryo quality. Therefore, E-EVs could be used in liquid biopsy for noninvasive genetic diagnosis.<\/jats:p>","DOI":"10.3390\/ani14071041","type":"journal-article","created":{"date-parts":[[2024,4,4]],"date-time":"2024-04-04T11:11:09Z","timestamp":1712229069000},"page":"1041","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["DNA Content in Embryonic Extracellular Vesicles Is Independent of the Apoptotic Rate in Bovine Embryos Produced In Vitro"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2772-8793","authenticated-orcid":false,"given":"Diego","family":"Caama\u00f1o","sequence":"first","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joel","family":"Cabezas","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Constanza","family":"Aguilera","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ioanna","family":"Martinez","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5126-6325","authenticated-orcid":false,"given":"Yat Sen","family":"Wong","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela Sanhueza","family":"Sagredo","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bel\u00e9n","family":"Iba\u00f1ez","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sebasti\u00e1n","family":"Rodriguez","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8828-1449","authenticated-orcid":false,"given":"Fidel Ovidio","family":"Castro","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4364-1097","authenticated-orcid":false,"given":"Lleretny","family":"Rodriguez-Alvarez","sequence":"additional","affiliation":[{"name":"Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepci\u00f3n, Av. Vicente Mendez 595, Chill\u00e1n 3780000, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,3,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1535750","DOI":"10.1080\/20013078.2018.1535750","article-title":"Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines","volume":"7","author":"Witwer","year":"2018","journal-title":"J. Extracell. Vesicles"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1093\/intimm\/dxh267","article-title":"Exosomal-like vesicles are present in human blood plasma","volume":"17","author":"Caby","year":"2005","journal-title":"Int. Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"13368","DOI":"10.1073\/pnas.0403453101","article-title":"Identification and proteomic profiling of exosomes in human urine","volume":"101","author":"Pisitkun","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1183","DOI":"10.1248\/bpb.b16-00891","article-title":"Characterization of Membrane Integrity and Morphological Stability of Human Salivary Exosomes","volume":"40","author":"Kumeda","year":"2017","journal-title":"Biol. Pharm. Bull."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1969","DOI":"10.4049\/jimmunol.179.3.1969","article-title":"Exosomes with immune modulatory features are present in human breast milk","volume":"179","author":"Admyre","year":"2007","journal-title":"J. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.jri.2008.06.001","article-title":"Heat shock protein-containing exosomes in mid-trimester amniotic fluids","volume":"79","author":"Asea","year":"2008","journal-title":"J. Reprod. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"9149","DOI":"10.1038\/s41598-021-88163-1","article-title":"Extracellular vesicles derived from ascitic fluid enhance growth and migration of ovarian cancer cells","volume":"11","author":"Mitra","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3210","DOI":"10.1242\/dev.124289","article-title":"Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome","volume":"142","author":"Vilella","year":"2015","journal-title":"Development"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"G990","DOI":"10.1152\/ajpgi.00093.2010","article-title":"Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia","volume":"299","author":"Masyuk","year":"2010","journal-title":"Am. J. Physiol. Gastrointest. Liver Physiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e0178306","DOI":"10.1371\/journal.pone.0178306","article-title":"Identification and characteristics of extracellular vesicles from bovine blastocysts produced in vitro","volume":"12","author":"Mellisho","year":"2017","journal-title":"PLoS ONE"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Battistelli, M., and Falcieri, E. (2020). Apoptotic Bodies: Particular Extracellular Vesicles Involved in Intercellular Communication. Biology, 9.","DOI":"10.3390\/biology9010021"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s11060-013-1084-8","article-title":"Biogenesis of extracellular vesicles (EV): Exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies","volume":"113","author":"Akers","year":"2013","journal-title":"J. Neurooncol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2667","DOI":"10.1007\/s00018-011-0689-3","article-title":"Membrane vesicles, current state-of-the-art: Emerging role of extracellular vesicles","volume":"68","author":"Aradi","year":"2011","journal-title":"Cell Mol. Life Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.tcb.2008.11.003","article-title":"Shedding microvesicles: Artefacts no more","volume":"19","author":"Cocucci","year":"2009","journal-title":"Trends Cell Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1795","DOI":"10.1002\/glia.22558","article-title":"Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system","volume":"61","author":"Picou","year":"2013","journal-title":"Glia"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"a037275","DOI":"10.1101\/cshperspect.a037275","article-title":"Extracellular Vesicles and Metastasis","volume":"10","author":"Wang","year":"2020","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"D516","DOI":"10.1093\/nar\/gky1029","article-title":"Vesiclepedia 2019: A compendium of RNA, proteins, lipids and metabolites in extracellular vesicles","volume":"47","author":"Pathan","year":"2019","journal-title":"Nucleic Acids Res"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1083\/jcb.201211138","article-title":"Extracellular vesicles: Exosomes, microvesicles, and friends","volume":"200","author":"Raposo","year":"2013","journal-title":"J. Cell Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1111\/ene.13439","article-title":"Alzheimer\u2019s disease","volume":"25","author":"Lane","year":"2018","journal-title":"Eur. J. Neurol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Tatischeff, I. (2023). Extracellular Vesicle-DNA: The Next Liquid Biopsy Biomarker for Early Cancer Diagnosis?. Cancers, 15.","DOI":"10.3390\/cancers15051456"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.theriogenology.2023.10.005","article-title":"Embryo-maternal communication mediated by extracellular vesicles in the early stages of embryonic development is modified by in vitro conditions","volume":"214","author":"Aguilera","year":"2024","journal-title":"Theriogenology"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Aguilera, C., Vel\u00e1squez, A.E., Gutierrez-Reinoso, M.A., Wong, Y.S., Melo-Baez, B., Cabezas, J., Caama\u00f1o, D., Navarrete, F., Rojas, D., and Riadi, G. (2023). Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24087438"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.theriogenology.2020.03.008","article-title":"Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers","volume":"149","author":"Dissanayake","year":"2020","journal-title":"Theriogenology"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Pavani, K.C., Hendrix, A., Van Den Broeck, W., Couck, L., Szymanska, K., Lin, X., De Koster, J., Van Soom, A., and Leemans, B. (2018). Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20010038"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1530\/REP-19-0233","article-title":"Extracellular vesicles secreted during blastulation show viability of bovine embryos","volume":"158","author":"Mellisho","year":"2019","journal-title":"Reproduction"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5210","DOI":"10.1038\/s41598-017-05549-w","article-title":"Secretome of in vitro cultured human embryos contains extracellular vesicles that are uptaken by the maternal side","volume":"7","author":"Giacomini","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"151125","DOI":"10.1016\/j.ejcb.2020.151125","article-title":"Extracellular vesicles: Mediators of embryo-maternal crosstalk during pregnancy and a new weapon to fight against infertility","volume":"99","author":"Das","year":"2020","journal-title":"Eur. J. Cell Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1089\/cell.2014.0003","article-title":"Improvement of cloned embryos development by co-culturing with parthenotes: A possible role of exosomes\/microvesicles for embryos paracrine communication","volume":"16","author":"Saadeldin","year":"2014","journal-title":"Cell Reprogram"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"39927","DOI":"10.1038\/srep39927","article-title":"A simple and rapid flow cytometry-based assay to identify a competent embryo prior to embryo transfer","volume":"7","author":"Pallinger","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/j.cell.2017.02.005","article-title":"Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency","volume":"169","author":"Yang","year":"2017","journal-title":"Cell"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"7193075","DOI":"10.1155\/2016\/7193075","article-title":"The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis","volume":"2016","author":"Cimadomo","year":"2016","journal-title":"Biomed. Res. Int."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1007\/s10815-019-01437-6","article-title":"Current status of spent embryo media research for preimplantation genetic testing","volume":"36","author":"Belandres","year":"2019","journal-title":"J. Assist Reprod Genet"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/j.fertnstert.2016.10.015","article-title":"Characterizing nuclear and mitochondrial DNA in spent embryo culture media: Genetic contamination identified","volume":"107","author":"Hammond","year":"2017","journal-title":"Fertil. Steril."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1701","DOI":"10.1002\/1878-0261.12777","article-title":"DNA in extracellular vesicles: Biological and clinical aspects","volume":"15","author":"Elzanowska","year":"2021","journal-title":"Mol. Oncol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1017\/S0967199414000677","article-title":"Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation","volume":"24","author":"Velasquez","year":"2016","journal-title":"Zygote"},{"key":"ref_36","first-page":"1","article-title":"Exosomal PD-L1 promotes tumor growth through immune escape in non-small cell lung cancer","volume":"51","author":"Kim","year":"2019","journal-title":"Exp. Mol. Med."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2137","DOI":"10.1007\/s43032-023-01166-3","article-title":"Haploid Parthenogenetic Embryos Exhibit Unique Stress Response to pH, Osmotic and Oxidative Stress","volume":"30","author":"Crasta","year":"2023","journal-title":"Reprod. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Melo-Baez, B., Wong, Y.S., Aguilera, C.J., Cabezas, J., Man\u00e7anares, A.C.F., Riadi, G., Castro, F.O., and Rodriguez-Alvarez, L. (2020). MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21238888"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"jcs222406","DOI":"10.1242\/jcs.222406","article-title":"New insights into extracellular vesicle biogenesis and function","volume":"132","author":"Latifkar","year":"2019","journal-title":"J. Cell Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3869","DOI":"10.1074\/jbc.C113.532267","article-title":"Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer","volume":"289","author":"Kahlert","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"15287","DOI":"10.1038\/ncomms15287","article-title":"Exosomes maintain cellular homeostasis by excreting harmful DNA from cells","volume":"8","author":"Takahashi","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1200\/JCO.2019.37.4_suppl.107","article-title":"The impact of muscle mass loss after esophagectomy for esophageal cancer on prognosis","volume":"37","author":"Shimoda","year":"2019","journal-title":"J. Clin. Oncol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1016\/j.cell.2014.09.051","article-title":"Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways","volume":"159","author":"Boelens","year":"2014","journal-title":"Cell"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1017\/S0967199420000593","article-title":"Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos","volume":"29","author":"Veraguas","year":"2021","journal-title":"Zygote"},{"key":"ref_45","first-page":"16","article-title":"Short Communication: Pre-implantation bovine embryos secrete small extracellular vesicles that contain embryonic DNA useful for genetic analysis","volume":"40","author":"Cabezas","year":"2022","journal-title":"Embryo Technol. Newsl."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"852620","DOI":"10.3389\/fendo.2022.852620","article-title":"Application of Two Blastocyst Biopsy Strategies in Preimplantation Genetic Testing Treatment and Assessment of Their Effects","volume":"13","author":"Yang","year":"2022","journal-title":"Front. Endocrinol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1016\/j.rbmo.2020.02.002","article-title":"Is cell-free DNA in spent embryo culture medium an alternative to embryo biopsy for preimplantation genetic testing? A systematic review","volume":"40","author":"Brouillet","year":"2020","journal-title":"Reprod. Biomed. Online"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2652","DOI":"10.1093\/humrep\/det314","article-title":"Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation","volume":"28","author":"Stigliani","year":"2013","journal-title":"Hum. Reprod."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1905","DOI":"10.2174\/092986709788186075","article-title":"Pertinence of apoptosis markers for the improvement of in vitro fertilization (IVF)","volume":"16","author":"Haouzi","year":"2009","journal-title":"Curr. Med. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1111\/j.1439-0531.2008.01131.x","article-title":"Influence of apoptosis in bovine embryo\u2019s development","volume":"45","author":"Antunes","year":"2010","journal-title":"Reprod. Domest. Anim."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1007\/s10815-011-9608-7","article-title":"Preimplantation genetic screening: Does it help or hinder IVF treatment and what is the role of the embryo?","volume":"28","author":"Ly","year":"2011","journal-title":"J. Assist. Reprod. Genet."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/14\/7\/1041\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:20:37Z","timestamp":1760106037000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/14\/7\/1041"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,3,29]]},"references-count":51,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["ani14071041"],"URL":"https:\/\/doi.org\/10.3390\/ani14071041","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,3,29]]}}}