{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:00:28Z","timestamp":1760234428113,"version":"build-2065373602"},"reference-count":56,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2021,5,20]],"date-time":"2021-05-20T00:00:00Z","timestamp":1621468800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31972547"],"award-info":[{"award-number":["31972547"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Key Research and Development Program","award":["2017YFE0108000"],"award-info":[{"award-number":["2017YFE0108000"]}]},{"name":"Qing Lan project of Jiangsu Province","award":["2020"],"award-info":[{"award-number":["2020"]}]},{"name":"Excellent Educational Talents Program of the Fourth &quot; Talent Cultivation Plan&quot; in Yangzhou City","award":["2020"],"award-info":[{"award-number":["2020"]}]},{"name":"Natural Science Project of Yangzhou Polytechnic College","award":["2018ZR19"],"award-info":[{"award-number":["2018ZR19"]}]},{"name":"Young Academic leaders project of Yangzhou Polytechnic College","award":["yzd2020-08"],"award-info":[{"award-number":["yzd2020-08"]}]},{"name":"Jiangsu Science and Technology Project (Youth Fund)","award":["BK20180918"],"award-info":[{"award-number":["BK20180918"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Alternative splicing (AS) is a ubiquitous, co-transcriptional, and post-transcriptional regulation mechanism during certain developmental processes, such as germ cell differentiation. A thorough understanding of germ cell differentiation will help us to open new avenues for avian reproduction, stem cell biology, and advances in medicines for human consumption. Here, based on single-cell RNA-seq, we characterized genome-wide AS events in manifold chicken male germ cells: embryonic stem cells (ESCs), gonad primordial germ cells (gPGCs), and spermatogonia stem cells (SSCs). A total of 38,494 AS events from 15,338 genes were detected in ESCs, with a total of 48,955 events from 14,783 genes and 49,900 events from 15,089 genes observed in gPGCs and SSCs, respectively. Moreover, this distribution of AS events suggests the diverse splicing feature of ESCs, gPGCs, and SSCs. Finally, several crucial stage-specific genes, such as NANOG, POU5F3, LIN28B, BMP4, STRA8, and LHX9, were identified in AS events that were transmitted in ESCs, gPGCs, and SSCs. The gene expression results of the RNA-seq data were validated by qRT-PCR. In summary, we provided a comprehensive atlas of the genome-wide scale of the AS event landscape in male chicken germ-line cells and presented its distribution for the first time. This research may someday improve treatment options for men suffering from male infertility.<\/jats:p>","DOI":"10.3390\/ani11051469","type":"journal-article","created":{"date-parts":[[2021,5,20]],"date-time":"2021-05-20T11:45:57Z","timestamp":1621511157000},"page":"1469","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Characterization of Alternative Splicing (AS) Events during Chicken (Gallus gallus) Male Germ-Line Stem Cell Differentiation with Single-Cell RNA-seq"],"prefix":"10.3390","volume":"11","author":[{"given":"Changhua","family":"Sun","sequence":"first","affiliation":[{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Department of Food Technology, College of Biochemical Engineering, Yangzhou Polytechnic College, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2489-2173","authenticated-orcid":false,"given":"Kai","family":"Jin","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qisheng","family":"Zuo","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hongyan","family":"Sun","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6973-5959","authenticated-orcid":false,"given":"Jiuzhou","family":"Song","sequence":"additional","affiliation":[{"name":"Animal &amp; Avian Sciences, University of Maryland, College Park, MD 20741, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yani","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guohong","family":"Chen","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bichun","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,5,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1038\/nature08909","article-title":"Expansion of the eukaryotic proteome by alternative splicing","volume":"463","author":"Nilsen","year":"2010","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1146\/annurev-biochem-060614-034316","article-title":"Mechanisms and regulation of alternative Pre-mRNA splicing","volume":"84","author":"Lee","year":"2015","journal-title":"Annu. Rev. Biochem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1038\/nrm2777","article-title":"Mechanisms of alternative splicing regulation: Insights from molecular and genomics approaches","volume":"10","author":"Chen","year":"2009","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1101\/gr.114645.110","article-title":"Genome-wide analysis of alternative splicing in caenorhabditis elegans","volume":"21","author":"Ramani","year":"2011","journal-title":"Genome Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1413","DOI":"10.1038\/ng.259","article-title":"Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing","volume":"40","author":"Pan","year":"2008","journal-title":"Nat. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Chacko, E., and Ranganathan, S. (2009). Genome-wide analysis of alternative splicing in cow: Implications in bovine as a model for human diseases. BMC Genom., 10.","DOI":"10.1186\/1471-2164-10-S3-S11"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1038\/nrg2776","article-title":"Alternative splicing and evolution: Diversification, exon definition and function","volume":"11","author":"Keren","year":"2010","journal-title":"Nat. Rev. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Yabas, M., Elliott, H., and Hoyne, G.F. (2016). The role of alternative splicing in the control of immune homeostasis and cellular differentiation. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17010003"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13059-018-1512-3","article-title":"Alternative splicing links histone modifications to stem cell fate decision","volume":"19","author":"Xu","year":"2018","journal-title":"Genome Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1175","DOI":"10.1007\/s00438-017-1350-0","article-title":"The determinants of alternative RNA splicing in human cells","volume":"292","author":"Ramanouskaya","year":"2017","journal-title":"Mol. Genet. Genom."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Fiszbein, A., and Kornblihtt, A.R. (2017). Alternative splicing switches: Important players in cell differentiation. Bioessays, 39.","DOI":"10.1002\/bies.201600157"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1007\/978-1-4939-4017-2_13","article-title":"Mouse fetal germ cell isolation and culture techniques","volume":"1463","author":"Spiller","year":"2017","journal-title":"Methods Mol. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.tig.2011.09.004","article-title":"De novo DNA methylation: A germ cell perspective","volume":"28","author":"Smallwood","year":"2012","journal-title":"Trends Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"a008375","DOI":"10.1101\/cshperspect.a008375","article-title":"Primordial germ cells in mice","volume":"4","author":"Saitou","year":"2012","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.scr.2018.03.015","article-title":"Production of live fish derived from frozen germ cells via germ cell transplantation","volume":"29","author":"Yoshizaki","year":"2018","journal-title":"Stem Cell Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-981-10-3975-1_1","article-title":"Avian primordial germ cells","volume":"1001","author":"Tagami","year":"2017","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_17","first-page":"121","article-title":"In vitro differentiation of primordial germ cells and oocyte-like cells from stem cells","volume":"33","author":"Costa","year":"2018","journal-title":"Histol. Histopathol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3113","DOI":"10.1242\/dev.118570","article-title":"Modeling mouse and human development using organoid cultures","volume":"142","author":"Huch","year":"2015","journal-title":"Development"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1007\/s12015-016-9661-9","article-title":"Multipotent differentiation of human dental pulp stem cells: A literature Review","volume":"12","author":"Nuti","year":"2016","journal-title":"Stem Cell Rev. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"681","DOI":"10.1080\/00071668.2017.1365354","article-title":"The evolution of chicken stem cell culture methods","volume":"58","author":"Farzaneh","year":"2017","journal-title":"Br. Poult. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1387\/ijdb.170283jh","article-title":"The early development of germ cells in chicken","volume":"62","author":"Kim","year":"2018","journal-title":"Int. J. Dev. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1159\/000103184","article-title":"The chicken as a model for embryonic development","volume":"117","author":"Davey","year":"2007","journal-title":"Cytogenet. Genome Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12864-018-4441-3","article-title":"Genetical genomics of growth in a chicken model","volume":"19","author":"Johnsson","year":"2018","journal-title":"BMC Genom."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1007\/s10456-014-9440-7","article-title":"The chicken chorioallantoic membrane model in biology, medicine and bioengineering","volume":"17","author":"Segura","year":"2014","journal-title":"Angiogenesis"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2598","DOI":"10.1016\/j.celrep.2017.05.089","article-title":"Ptbp2 controls an alternative splicing network required for cell communication during spermatogenesis","volume":"19","author":"Hannigan","year":"2017","journal-title":"Cell Rep."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4030","DOI":"10.1128\/MCB.00676-15","article-title":"RNA binding protein Ptbp2 is essential for male germ cell development","volume":"35","author":"Zagore","year":"2015","journal-title":"Mol. Cell Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1038\/s41422-018-0074-y","article-title":"Single-Cell RNA-Seq uncovers dynamic processes and critical regulators in mouse spermatogenesis","volume":"28","author":"Chen","year":"2018","journal-title":"Cell Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1101\/gr.161034.113","article-title":"From single-cell to cell-pool transcriptomes: Stochasticity in gene expression and RNA splicing","volume":"24","author":"Marinov","year":"2014","journal-title":"Genome Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"317","DOI":"10.3389\/fgene.2019.00317","article-title":"Single-cell RNA-Seq technologies and related computational data analysis","volume":"10","author":"Chen","year":"2019","journal-title":"Front. Genet."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"13605","DOI":"10.1074\/jbc.M114.601401","article-title":"Crucial genes and pathways in chicken germ stem cell differentiation","volume":"290","author":"Zhang","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Gao, W., Zhang, C., Jin, K., Zhang, Y., Zuo, Q., and Li, B. (2020). Analysis of LncRNA expression profile during the formation of male germ cells in chickens. Animals, 10.","DOI":"10.3390\/ani10101850"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"R36","DOI":"10.1186\/gb-2013-14-4-r36","article-title":"TopHat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions","volume":"14","author":"Kim","year":"2013","journal-title":"Genome Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1038\/nprot.2012.016","article-title":"Differential gene and transcript expression analysis of RNA-Seq experiments with TopHat and cufflinks","volume":"7","author":"Trapnell","year":"2012","journal-title":"Nat. Protoc."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"188","DOI":"10.12688\/f1000research.2-188.v1","article-title":"Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues","volume":"2","author":"Florea","year":"2013","journal-title":"F1000Research"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/nar\/gkn923","article-title":"Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists","volume":"37","author":"Huang","year":"2009","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1038\/nprot.2008.211","article-title":"Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources","volume":"4","author":"Huang","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3448","DOI":"10.1093\/bioinformatics\/bti551","article-title":"BiNGO: A cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks","volume":"21","author":"Maere","year":"2005","journal-title":"Bioinformatics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"W720","DOI":"10.1093\/nar\/gkl167","article-title":"KOBAS server: A web-based platform for automated annotation and pathway identification","volume":"34","author":"Wu","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1016\/j.bbagrm.2016.03.013","article-title":"Evolution and functions of Oct4 homologs in non-mammalian vertebrates","volume":"1859","author":"Onichtchouk","year":"2016","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1700086","DOI":"10.1002\/bies.201700086","article-title":"Nanog expression in embryonic stem cells\u2014An ideal model system to dissect enhancer function","volume":"39","author":"Blinka","year":"2017","journal-title":"Bioessays"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1101\/gad.13.4.424","article-title":"Bmp4 is required for the generation of primordial germ cells in the mouse embryo","volume":"13","author":"Lawson","year":"1999","journal-title":"Genes Dev."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1038\/nature08210","article-title":"A role for Lin28 in primordial germ-cell development and germ-cell malignancy","volume":"460","author":"West","year":"2009","journal-title":"Nature"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1497","DOI":"10.1007\/s00429-014-0740-x","article-title":"The Lhx9 homeobox gene controls pineal gland development and prevents postnatal hydrocephalus","volume":"220","author":"Yamazaki","year":"2015","journal-title":"Brain Struct. Funct."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Wang, Y., and Zuo, Q. (2017). CRISPR\/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0172207"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1038\/nrm.2017.27","article-title":"Alternative splicing as a regulator of development and tissue identity","volume":"18","author":"Baralle","year":"2017","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1258","DOI":"10.1016\/j.drudis.2019.03.030","article-title":"Alternative splicing, RNA-Seq and drug discovery","volume":"24","author":"Zhao","year":"2019","journal-title":"Drug Discov. Today"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2987","DOI":"10.1002\/1873-3468.13119","article-title":"Regulation of alternative MRNA splicing: Old players and new perspectives","volume":"592","author":"Dvinge","year":"2018","journal-title":"FEBS Lett."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"38","DOI":"10.7150\/ijbs.34422","article-title":"The function of Pre-MRNA alternative splicing in mammal spermatogenesis","volume":"16","author":"Song","year":"2020","journal-title":"Int. J. Biol. Sci."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Guan, Y., Liang, G., Martin, G.B., and Guan, L.L. (2017). Functional changes in MRNA expression and alternative Pre-MRNA splicing associated with the effects of nutrition on apoptosis and spermatogenesis in the adult testis. BMC Genom., 18.","DOI":"10.1186\/s12864-016-3385-8"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.gep.2013.10.002","article-title":"Dynamic expression of combinatorial replication-dependent histone variant genes during mouse spermatogenesis","volume":"14","author":"Sun","year":"2014","journal-title":"Gene Expr. Patterns"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1016\/j.devcel.2020.01.014","article-title":"Metabolic changes accompanying spermatogonial stem cell differentiation","volume":"52","author":"Lord","year":"2020","journal-title":"Dev. Cell"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1111\/j.1749-6632.1980.tb15971.x","article-title":"Calcium explosions as triggers of development","volume":"339","author":"Jaffe","year":"1980","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"051001","DOI":"10.1088\/1478-3975\/aabb18","article-title":"Calcium as a signal integrator in developing epithelial tissues","volume":"15","author":"Brodskiy","year":"2018","journal-title":"Phys. Biol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"34510","DOI":"10.1038\/srep34510","article-title":"Wnt\/\u03b2-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro","volume":"6","author":"Lee","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"14703","DOI":"10.1096\/fj.201900782R","article-title":"SIRT1 facilitates primordial follicle recruitment independent of deacetylase activity through directly modulating Akt1 and MTOR transcription","volume":"33","author":"Zhang","year":"2019","journal-title":"FASEB J."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.diff.2014.06.001","article-title":"An in vitro study of the impact of 4mT static magnetic field to modify the differentiation rate of rat bone marrow stem cells into primordial germ cells","volume":"87","author":"Jouni","year":"2014","journal-title":"Differentiation"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/11\/5\/1469\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:04:42Z","timestamp":1760162682000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/11\/5\/1469"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,5,20]]},"references-count":56,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["ani11051469"],"URL":"https:\/\/doi.org\/10.3390\/ani11051469","relation":{},"ISSN":["2076-2615"],"issn-type":[{"type":"electronic","value":"2076-2615"}],"subject":[],"published":{"date-parts":[[2021,5,20]]}}}