{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T03:46:30Z","timestamp":1790394390698,"version":"4.1.0"},"reference-count":115,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T00:00:00Z","timestamp":1741737600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Vet. Sci."],"abstract":"<jats:p>Sex determination is the developmental assignment that results from genetic factors. The sexual characters were the specific manifestations of male and female individuals under stimulation of sexual hormonal production. The fusion of an oocyte with an X chromosome-bearing sperm will lead to a female (XX), while fusion with a Y chromosome-bearing sperm will develop into a male (XY) in mammals. Sexing technology has been developed to fertilize eggs with sorted sperm, producing offspring of the desired sex. Sperm sorting enables the sex pre-determination of offspring via <jats:italic>in vitro<\/jats:italic> fertilization (IVF) or artificial insemination (AI) in domestic animals. Flow cytometric sorting of X and Y sperm is widely considered the most applied method for sperm sorting and has been commercially applied in cattle. However, a non-invasive, immunological method for screening X and Y sperm is considered to be a feasible approach. This review summarizes the current knowledge and techniques of sperm immunological sorting, including the preparation of antibodies, application of immunomodulators, and immunoisolation. Additionally, we focus on identifying sex-specifically expressed proteins in X and Y sperm through proteomic analysis, and verifying the sex-specific proteins using experimental techniques. Furthermore, several housekeeping proteins as loading control were discussed in immunoblotting of sperm proteins. Immunological sorting of X and Y sperm could provide a convenient, cost-effective, and highly efficient technique that can improve economic benefits and achieve an advanced level of sexing technology. This review provides insight into immunological sorting of sperm and the pre-determination of sex in farm animals.<\/jats:p>","DOI":"10.3389\/fvets.2025.1523491","type":"journal-article","created":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T07:55:55Z","timestamp":1741766155000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":10,"title":["Advances in immunological sorting of X and Y chromosome-bearing sperm: from proteome to sex-specific proteins"],"prefix":"10.3389","volume":"12","author":[{"given":"Linfeng","family":"Bai","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongli","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gaoping","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhigang","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xihe","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,3,12]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"e1900079","DOI":"10.1002\/adbi.201900079","article-title":"Review on sperm sorting technologies and sperm properties toward new separation methods via the Interface of biochemistry and material science","volume":"3","author":"Katigbak","year":"2019","journal-title":"Adv Biosyst"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1016\/j.theriogenology.2019.05.039","article-title":"Pre-determination of sex in pigs by application of Crispr\/Cas system for genome editing","volume":"137","author":"Kurtz","year":"2019","journal-title":"Theriogenology"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"388","DOI":"10.3389\/fcell.2019.00388","article-title":"New biological insights on X and Y chromosome-bearing spermatozoa","volume":"7","author":"Rahman","year":"2019","journal-title":"Front Cell Dev Biol"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1016\/j.theriogenology.2020.12.028","article-title":"Small ruminant Sexedultra\u2122 sperm sex-sorting: status report and recent developments","volume":"162","author":"Gonz\u00e1lez-Mar\u00edn","year":"2021","journal-title":"Theriogenology"},{"key":"ref5","doi-asserted-by":"publisher","first-page":"279","DOI":"10.1007\/978-1-62703-038-0_26","article-title":"Sex-sorting sperm using flow cytometry\/cell sorting","volume":"927","author":"Garner","year":"2013","journal-title":"Methods Mol Biol"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"21745","DOI":"10.1021\/acsomega.3c01287","article-title":"Immunomagnetic isolation of Her2-positive breast Cancer cells using a microfluidic device","volume":"8","author":"Parvin","year":"2023","journal-title":"ACS Omega"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1016\/j.addr.2007.08.034","article-title":"Challenges and emerging Technologies in the Immunoisolation of cells and tissues","volume":"60","author":"Wilson","year":"2008","journal-title":"Adv Drug Deliv Rev"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"204","DOI":"10.3389\/fcimb.2020.00204","article-title":"Review on the current trends of toxoplasmosis Serodiagnosis in humans","volume":"10","author":"Yba\u00f1ez","year":"2020","journal-title":"Front Cell Infect Microbiol"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"231","DOI":"10.1093\/humrep\/15.2.231","article-title":"Antisperm antibodies: use of the mixed agglutination reaction (mar) test using latex beads","volume":"15","author":"Mahmoud","year":"2000","journal-title":"Hum Reprod"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1046\/j.1526-0968.2001.00370.x","article-title":"Background and indications for protein a-based extracorporeal Immunoadsorption","volume":"5","author":"Matic","year":"2001","journal-title":"Ther Apher"},{"key":"ref11","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/j.smim.2018.02.003","article-title":"Targeting complement-mediated Immunoregulation for Cancer immunotherapy","volume":"37","author":"Kolev","year":"2018","journal-title":"Semin Immunol"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"593610","DOI":"10.3389\/fimmu.2020.593610","article-title":"How does complement affect hematological malignancies: from basic mechanisms to clinical application","volume":"11","author":"Luo","year":"2020","journal-title":"Front Immunol"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.molimm.2022.08.017","article-title":"The impact of human complement on the clinical outcome of malaria infection","volume":"151","author":"Reiss","year":"2022","journal-title":"Mol Immunol"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"100486","DOI":"10.1016\/j.repbio.2021.100486","article-title":"Spermatological parameters of immunologically sexed bull semen assessed by imaging flow cytometry, and dairy farm trial","volume":"21","author":"Thongkham","year":"2021","journal-title":"Reprod Biol"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"508","DOI":"10.1080\/10495398.2020.1811294","article-title":"Production of single-chain fragment variable (Scfv) antibodies specific to plasma membrane epitopes on bull Y-bearing sperm","volume":"33","author":"Thaworn","year":"2022","journal-title":"Anim Biotechnol"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"715","DOI":"10.3390\/biology11050715","article-title":"High-efficiency bovine sperm sexing used magnetic-activated cell sorting by coupling Scfv antibodies specific to Y-chromosome-bearing sperm on magnetic microbeads","volume":"11","author":"Sringarm","year":"2022","journal-title":"Biology"},{"key":"ref17","year":"2017"},{"key":"ref18","unstructured":"2023"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"308","DOI":"10.1038\/246308a0","article-title":"Sex ratio in progeny of mice inseminated with sperm treated with H-Y antiserum","volume":"246","author":"Bennett","year":"1973","journal-title":"Nature"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1097\/00007890-198401000-00004","article-title":"Male-restricted expression of H-Y antigen on preimplantation mouse embryos","volume":"37","author":"Shelton","year":"1984","journal-title":"Transplantation"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"231","DOI":"10.1292\/jvms.12-0303","article-title":"Application of a new convenience gender sorting method for mouse spermatozoa to mouse reproductive engineering technology","volume":"75","author":"Hashimoto","year":"2013","journal-title":"J Vet Med Sci"},{"key":"ref22","first-page":"326","article-title":"Goat polyclonal antibody against the sex determining region Y to separate X- and Y-chromosome bearing spermatozoa","volume":"8","author":"Soleymani","year":"2019","journal-title":"Rep Biochem Mol Biol"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1111\/rda.13821","article-title":"Production of monoclonal antibody against recombinant bovine sex-determining region Y (Sry) and their preferential binding to Y chromosome-bearing sperm","volume":"56","author":"Soleymani","year":"2021","journal-title":"Reprod Domest Anim"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1016\/j.anireprosci.2018.09.010","article-title":"Proteomic profile of sex-sorted bull sperm evaluated by swath-Ms analysis","volume":"198","author":"Scott","year":"2018","journal-title":"Anim Reprod Sci"},{"key":"ref25","doi-asserted-by":"publisher","first-page":"181","DOI":"10.1016\/j.rvsc.2021.11.013","article-title":"Differential proteomic profile of X- and Y- sorted Sahiwal bull semen","volume":"144","author":"Sharma","year":"2022","journal-title":"Res Vet Sci"},{"key":"ref26","doi-asserted-by":"publisher","first-page":"3031","DOI":"10.1021\/acs.jproteome.0c00358","article-title":"Differential membrane protein profile in bovine X- and Y-sperm","volume":"20","author":"Shen","year":"2021","journal-title":"J Proteome Res"},{"key":"ref27","doi-asserted-by":"publisher","first-page":"e3000398","DOI":"10.1371\/journal.pbio.3000398","article-title":"Activation of toll-like receptor 7\/8 encoded by the X chromosome alters sperm motility and provides a novel simple Technology for Sexing Sperm","volume":"17","author":"Umehara","year":"2019","journal-title":"PLoS Biol"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"2645","DOI":"10.1038\/s41596-020-0348-y","article-title":"A simple sperm-sexing method that activates Tlr7\/8 on X sperm for the efficient production of sexed mouse or cattle embryos","volume":"15","author":"Umehara","year":"2020","journal-title":"Nat Protoc"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"426","DOI":"10.3390\/mi13030426","article-title":"Antibody-conjugated magnetic beads for sperm sexing using a Multi-Wall carbon nanotube microfluidic device","volume":"13","author":"Phiphattanaphiphop","year":"2022","journal-title":"Micromachines"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"156","DOI":"10.1016\/j.anireprosci.2018.11.006","article-title":"In vitro production of sex preselected cattle embryos using a monoclonal antibody raised against bull sperm epitopes","volume":"205","author":"Chowdhury","year":"2019","journal-title":"Anim Reprod Sci"},{"key":"ref31","doi-asserted-by":"publisher","first-page":"244","DOI":"10.3390\/ijms21010244","article-title":"Difference in developmental kinetics of Y-specific monoclonal antibody sorted male and female in vitro produced bovine embryos","volume":"21","author":"Sidrat","year":"2019","journal-title":"Int J Mol Sci"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"652","DOI":"10.3181\/00379727-85-20982","article-title":"Sperm agglutinins in human semen and blood","volume":"85","author":"Wilson","year":"1954","journal-title":"Proc Soc Exp Biol Med Soc Exp Biol Med"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1093\/ajcp\/32.4.357","article-title":"Autoantibodies against Spermatozoa in Sterile Men","volume":"32","author":"Rumke","year":"1959","journal-title":"Am J Clin Pathol"},{"key":"ref34","doi-asserted-by":"publisher","first-page":"677","DOI":"10.1016\/0002-9378(68)90307-4","article-title":"Immunologic analysis of sperm-immobilizing factor found in sera of women with unexplained sterility","volume":"101","author":"Isojima","year":"1968","journal-title":"Gynecology"},{"key":"ref35","doi-asserted-by":"publisher","first-page":"199","DOI":"10.1016\/0002-9378(72)90116-0","article-title":"Further studies on sperm-immobilizing antibody found in sera of unexplained cases of sterility in women","volume":"112","author":"Isojima","year":"1972","journal-title":"Am J Obstet Gynecol"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"335","DOI":"10.1016\/S0015-0282(16)41726-7","article-title":"The sperm-cervical mucus contact test: a preliminary report","volume":"27","author":"Kremer","year":"1976","journal-title":"Fertil Steril"},{"key":"ref37","first-page":"12","article-title":"Immune sperm agglutination: are only motile spermatozoa involved?","volume":"31","author":"Hellema","year":"1978","journal-title":"Clin Exp Immunol"},{"key":"ref38","first-page":"18","article-title":"Sperm autoantibodies as a consequence of vasectomy. I. Within 1 year post-operation","volume":"31","author":"Hellema","year":"1978","journal-title":"Clin Exp Immunol"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"502","DOI":"10.1016\/S0015-0282(98)00506-8","article-title":"Characterization of a potent sperm-agglutinating monoclonal antibody and its cognate antigens","volume":"71","author":"Yakirevich","year":"1999","journal-title":"Fertil Steril"},{"key":"ref40","first-page":"256","article-title":"Microtechnique for simultaneous determination of immobilizing and cytotoxic sperm antibodies. Methodological and clinical studies","volume":"22","author":"Husted","year":"1975","journal-title":"Clin Exp Immunol"},{"key":"ref41","doi-asserted-by":"publisher","first-page":"495","DOI":"10.1038\/256495a0","article-title":"Continuous cultures of fused cells secreting antibody of predefined specificity","volume":"256","author":"K\u00f6hler","year":"1975","journal-title":"Nature"},{"key":"ref42","first-page":"458","article-title":"Sperm-immobilizing monoclonal antibody to human seminal plasma antigens","volume":"42","author":"Shigeta","year":"1980","journal-title":"Clin Exp Immunol"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"S38","DOI":"10.1016\/0165-0378(81)90122-4","article-title":"Purification of human seminal plasma no. 7 antigen by Immunoaffinity chromatography on bound monoclonal antibody","volume":"2","author":"Isojima","year":"1982","journal-title":"Clin Exp Immunol"},{"key":"ref44","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1016\/0165-0378(82)90035-3","article-title":"Monoclonal antibodies to human sperm antigens","volume":"4","author":"Lee","year":"1982","journal-title":"J Reprod Immunol"},{"key":"ref45","doi-asserted-by":"publisher","first-page":"1015","DOI":"10.1095\/biolreprod30.4.1015","article-title":"Interspecies cross-reactivity of monoclonal antibodies directed against human sperm antigens","volume":"30","author":"Isahakia","year":"1984","journal-title":"Biol Reprod"},{"key":"ref46","first-page":"489","article-title":"A human-mouse Hybridoma producing monoclonal antibody against human sperm coating antigen","volume":"57","author":"Kyurkchiev","year":"1986","journal-title":"Immunology"},{"key":"ref47","doi-asserted-by":"publisher","first-page":"275","DOI":"10.1016\/0165-0378(86)90029-X","article-title":"Developmental studies of sperm surface antigens using sperm-specific monoclonal antibodies","volume":"9","author":"Lee","year":"1986","journal-title":"J Reprod Immunol"},{"key":"ref48","doi-asserted-by":"publisher","first-page":"567","DOI":"10.1002\/j.1939-4640.1996.tb01834.x","article-title":"Anti-bull sperm monoclonal antibodies: I. Identification of major antigenic domains of bull sperm and manifestation of interspecies cross-reactivity","volume":"17","author":"Ambrose","year":"1996","journal-title":"J Androl"},{"key":"ref49","doi-asserted-by":"publisher","first-page":"579","DOI":"10.1002\/j.1939-4640.1996.tb01835.x","article-title":"Anti-bull sperm monoclonal antibodies: ii. Binding changes during capacitation and influence on sperm-zona interactions in vitro","volume":"17","author":"Ambrose","year":"1996","journal-title":"J Androl"},{"key":"ref50","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1111\/j.1600-0897.1997.tb00231.x","article-title":"Monoclonal antibodies as direct probes for human sperm acrosome reaction","volume":"37","author":"Dorjee","year":"1997","journal-title":"Am J Reprod Immunol"},{"key":"ref51","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1111\/j.1600-0897.1987.tb00081.x","article-title":"Sex difference of antifertility effect by passively immunized monoclonal sperm antibodies","volume":"13","author":"Lee","year":"1987","journal-title":"Am J Reprod Immunol Microbiol"},{"key":"ref52","doi-asserted-by":"publisher","first-page":"1309","DOI":"10.1016\/S0093-691X(99)00219-8","article-title":"A new approach to immunological sexing of sperm","volume":"52","author":"Blecher","year":"1999","journal-title":"Theriogenology"},{"key":"ref53","first-page":"22","article-title":"Sumretprasong production of monoclonal antibody against male specific antigen on cell membrane of bovine spermatozoa","volume":"44","author":"Pattanawong","year":"2010","journal-title":"Indian J Anim Res"},{"key":"ref54","first-page":"425","article-title":"Monoclonal antibodies against male-specific antigen of white Lamphun cattle (Bos indicus)","volume":"48","author":"Dumrongsri","year":"2014","journal-title":"Kasetsart J (Nat Sci)"},{"key":"ref55","doi-asserted-by":"publisher","first-page":"878","DOI":"10.56093\/ijans.v86i8.60764","article-title":"Determination of male-specific gene expression of white Lamphun cattle semen treated by monoclonal antibodies","volume":"86","author":"Dumrongsri","year":"2016","journal-title":"Indian J Anim Sci"},{"key":"ref56","first-page":"148","article-title":"Skin","volume":"2","author":"Eichwald","year":"1955","journal-title":"Transpl Bullet"},{"key":"ref57","doi-asserted-by":"publisher","first-page":"1179","DOI":"10.1089\/scd.2019.0299","article-title":"The H-Y antigen in embryonic stem cells causes rejection in syngeneic female recipients","volume":"29","author":"Hu","year":"2020","journal-title":"Stem Cells Dev"},{"key":"ref58","doi-asserted-by":"publisher","first-page":"478","DOI":"10.1038\/232478a0","article-title":"Serological demonstration of H-Y (male) antigen on mouse sperm","volume":"232","author":"Goldberg","year":"1971","journal-title":"Nature"},{"key":"ref59","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1002\/mrd.1120200312","article-title":"Selection and separation of X- and Y- chromosome-bearing mammalian sperm","volume":"20","author":"Gledhill","year":"1988","journal-title":"Gamete Res"},{"key":"ref60","doi-asserted-by":"publisher","first-page":"3372","DOI":"10.3168\/jds.S0022-0302(89)79500-X","article-title":"Immunological sexing of mammalian semen: current status and future options","volume":"72","author":"Bradley","year":"1989","journal-title":"J Dairy Sci"},{"key":"ref61","doi-asserted-by":"publisher","first-page":"552","DOI":"10.1038\/312552a0","article-title":"Male sexual differentiation in mice lacking H-Y antigen","volume":"312","author":"McLaren","year":"1984","journal-title":"Nature"},{"key":"ref62","doi-asserted-by":"publisher","first-page":"235","DOI":"10.3109\/01485019008987580","article-title":"Enrichment of bovine X- and Y-chromosome-bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter","volume":"24","author":"Ali","year":"1990","journal-title":"Arch Androl"},{"key":"ref63","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1002\/mrd.1080350213","article-title":"Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome-bearing porcine and bovine sperm","volume":"35","author":"Hendriksen","year":"1993","journal-title":"Mol Reprod Dev"},{"key":"ref64","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1111\/j.1600-0897.1998.tb00387.x","article-title":"H-Y antigen expression patterns in human X- and Y-chromosome-bearing spermatozoa","volume":"40","author":"Sills","year":"1998","journal-title":"Am J Reprod Immunol"},{"key":"ref65","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1038\/346240a0","article-title":"A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif","volume":"346","author":"Sinclair","year":"1990","journal-title":"Nature"},{"key":"ref66","doi-asserted-by":"publisher","first-page":"245","DOI":"10.1038\/346245a0","article-title":"A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes","volume":"346","author":"Gubbay","year":"1990","journal-title":"Nature"},{"key":"ref67","doi-asserted-by":"publisher","first-page":"448","DOI":"10.1038\/348448a0","article-title":"Genetic evidence equating Sry and the testis-determining factor","volume":"348","author":"Berta","year":"1990","journal-title":"Nature"},{"key":"ref68","doi-asserted-by":"publisher","first-page":"450","DOI":"10.1038\/348450a0","article-title":"Expression of a candidate sex-determining gene during mouse testis differentiation","volume":"348","author":"Koopman","year":"1990","journal-title":"Nature"},{"key":"ref69","doi-asserted-by":"publisher","first-page":"4497","DOI":"10.1002\/j.1460-2075.1992.tb05551.x","article-title":"Sry, like Hmg1, recognizes sharp angles in DNA","volume":"11","author":"Ferrari","year":"1992","journal-title":"EMBO J"},{"key":"ref70","doi-asserted-by":"publisher","first-page":"6115","DOI":"10.1002\/j.1460-2075.1994.tb06958.x","article-title":"Sex-reversing mutations affect the architecture of Sry-DNA complexes","volume":"13","author":"Pontiggia","year":"1994","journal-title":"EMBO J"},{"key":"ref71","doi-asserted-by":"publisher","first-page":"930","DOI":"10.1038\/nature06944","article-title":"Sex determination involves synergistic action of Sry and Sf1 on a specific Sox9 enhancer","volume":"453","author":"Sekido","year":"2008","journal-title":"Nature"},{"key":"ref72","doi-asserted-by":"publisher","first-page":"1002","DOI":"10.1111\/j.1742-4658.2011.08029.x","article-title":"Sex determination and the control of Sox9 expression in mammals","volume":"278","author":"Jakob","year":"2011","journal-title":"FEBS J"},{"key":"ref73","first-page":"135","article-title":"The human Sry protein is present in fetal and adult Sertoli cells and germ cells","volume":"43","author":"Salas-Cort\u00e9s","year":"1999","journal-title":"Int J Dev Biol"},{"key":"ref74","doi-asserted-by":"publisher","first-page":"3324","DOI":"10.1002\/dvdy.22452","article-title":"A study of goat Sry protein expression suggests putative new roles for this gene in the developing testis of a species with Long-lasting Sry expression","volume":"239","author":"Montazer-Torbati","year":"2010","journal-title":"Dev Dynam"},{"key":"ref75","doi-asserted-by":"publisher","first-page":"1358","DOI":"10.5713\/ajas.2011.11062","article-title":"Detection of the Sry Transcript and Protein in Bovine Ejaculated Spermatozoa","volume":"24","author":"Li","year":"2011","journal-title":"Anim Biosci"},{"key":"ref76","doi-asserted-by":"publisher","first-page":"1323","DOI":"10.1242\/dev.161323","article-title":"Dynamic cytoplasmic projections connect mammalian Spermatogonia in vivo","volume":"145","author":"Niedenberger","year":"2018","journal-title":"Development"},{"key":"ref77","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1038\/337373a0","article-title":"Genetically haploid spermatids are phenotypically diploid","volume":"337","author":"Braun","year":"1989","journal-title":"Nature"},{"key":"ref78","doi-asserted-by":"publisher","first-page":"1723","DOI":"10.1126\/science.abb1723","article-title":"Widespread haploid-biased gene expression enables sperm-level natural selection","volume":"371","author":"Bhutani","year":"2021","journal-title":"Science"},{"key":"ref79","doi-asserted-by":"publisher","first-page":"100462","DOI":"10.1016\/j.xgen.2023.100462","article-title":"The human Y and inactive X chromosomes similarly modulate autosomal gene expression","volume":"4","author":"San Roman","year":"2024","journal-title":"Cell Genom"},{"key":"ref80","doi-asserted-by":"publisher","first-page":"6926","DOI":"10.1038\/s41467-021-27227-2","article-title":"Crispr-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes","volume":"12","author":"Douglas","year":"2021","journal-title":"Nat Commun"},{"key":"ref81","doi-asserted-by":"publisher","first-page":"195","DOI":"10.1530\/jrf.0.1100195","article-title":"A search for sex-specific antigens on bovine spermatozoa using immunological and biochemical techniques to compare the protein profiles of X and Y chromosome-bearing sperm populations separated by fluorescence-activated cell sorting","volume":"110","author":"Howes","year":"1997","journal-title":"J Reprod Fertil"},{"key":"ref82","doi-asserted-by":"publisher","first-page":"1633","DOI":"10.1002\/vms3.540","article-title":"Bovine semen sexing: sperm membrane proteomics as candidates for immunological selection of X- and Y-chromosome-bearing sperm","volume":"7","author":"Quelhas","year":"2021","journal-title":"Vet Med Sci"},{"key":"ref83","doi-asserted-by":"publisher","first-page":"2060","DOI":"10.3168\/jds.2010-3350","article-title":"An immunological method to screen sex-specific proteins of bovine sperm","volume":"94","author":"Sang","year":"2011","journal-title":"J Dairy Sci"},{"key":"ref84","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1007\/s11033-013-2854-2","article-title":"Tentative identification of sex-specific antibodies and their application for screening bovine sperm proteins for sex-specificity","volume":"41","author":"Yang","year":"2014","journal-title":"Mol Biol Rep"},{"key":"ref85","doi-asserted-by":"publisher","first-page":"59","DOI":"10.1016\/j.jprot.2012.07.004","article-title":"Identification of differentially expressed proteins between bull X and Y spermatozoa","volume":"77","author":"Chen","year":"2012","journal-title":"J Proteome"},{"key":"ref86","doi-asserted-by":"publisher","first-page":"1264","DOI":"10.1039\/C3MB70306A","article-title":"Differential protein profile in sexed bovine semen: shotgun proteomics investigation","volume":"10","author":"De Canio","year":"2014","journal-title":"Mol BioSyst"},{"key":"ref87","doi-asserted-by":"publisher","first-page":"3559","DOI":"10.1002\/pmic.201200133","article-title":"Comprehensive mapping of the bull sperm surface proteome","volume":"12","author":"Byrne","year":"2012","journal-title":"Proteomics"},{"key":"ref88","doi-asserted-by":"publisher","first-page":"1906","DOI":"10.1002\/pmic.201400297","article-title":"Bovine sperm plasma membrane proteomics through Biotinylation and subcellular enrichment","volume":"15","author":"Kasvandik","year":"2015","journal-title":"Proteomics"},{"key":"ref89","doi-asserted-by":"publisher","first-page":"3672","DOI":"10.3390\/ani14243672","article-title":"Proteomic and Metabolomic profiling reveals alterations in boar X and Y sperm","volume":"14","author":"Cheng","year":"2024","journal-title":"Animals"},{"key":"ref90","doi-asserted-by":"publisher","first-page":"498","DOI":"10.14202\/vetworld.2017.498-504","article-title":"An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm","volume":"10","author":"Yadav","year":"2017","journal-title":"Vet World"},{"key":"ref91","doi-asserted-by":"publisher","first-page":"423","DOI":"10.1017\/S0967199421000939","article-title":"Advancements in mammalian X and Y sperm differences and sex control technology","volume":"30","author":"He","year":"2022","journal-title":"Zygote"},{"key":"ref92","doi-asserted-by":"publisher","first-page":"e2207733","DOI":"10.1002\/adma.202207733","article-title":"Azide-masked Resiquimod activated by hypoxia for selective tumor therapy","volume":"35","author":"Sun","year":"2023","journal-title":"Adv Mater"},{"key":"ref93","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1358\/dnp.2008.21.2.1188193","article-title":"The antiviral activity of toll-like receptor 7 and 7\/8 agonists","volume":"21","author":"Miller","year":"2008","journal-title":"Drug News Perspect"},{"key":"ref94","doi-asserted-by":"publisher","first-page":"142","DOI":"10.3390\/pharmaceutics13020142","article-title":"Tlr agonists as vaccine adjuvants targeting Cancer and infectious diseases","volume":"13","author":"Luchner","year":"2021","journal-title":"Pharmaceutics"},{"key":"ref95","doi-asserted-by":"publisher","first-page":"123485","DOI":"10.1016\/j.ijbiomac.2023.123485","article-title":"Tlr7\/8 agonist (R848) inhibit bovine X sperm motility via Pi3k\/Gsk3\u03b1\/\u0392 and Pi3k\/Nf\u03bab pathways","volume":"232","author":"Wen","year":"2023","journal-title":"Int J Biol Macromol"},{"key":"ref96","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1186\/s40104-021-00613-y","article-title":"Tlr7\/8 Signalling affects X-sperm motility via the Gsk3 \u0391\/\u0392-hexokinase pathway for the efficient production of sexed dairy goat embryos","volume":"12","author":"Ren","year":"2021","journal-title":"J Anim Sci Biotechnol"},{"key":"ref97","doi-asserted-by":"publisher","first-page":"10020","DOI":"10.3168\/jds.2022-22115","article-title":"Alkaline semen diluent combined with R848 for separation and enrichment of dairy goat X-sperm","volume":"105","author":"Huang","year":"2022","journal-title":"J Dairy Sci"},{"key":"ref98","doi-asserted-by":"publisher","first-page":"747722","DOI":"10.3389\/fcell.2021.747722","article-title":"Effects of diluent Ph on enrichment and performance of dairy goat X\/Y sperm","volume":"9","author":"He","year":"2021","journal-title":"Front Cell Dev Biol"},{"key":"ref99","doi-asserted-by":"publisher","first-page":"1771","DOI":"10.3390\/ijms24021771","article-title":"Alkaline dilution alters sperm motility in dairy goat by affecting sac\/camp\/Pka pathway activity","volume":"24","author":"He","year":"2023","journal-title":"Int J Mol Sci"},{"key":"ref100","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.theriogenology.2023.02.012","article-title":"Diluent Ph affects sperm motility via Gsk3 \u0391\/\u0392-hexokinase pathway for the efficient enrichment of X-sperm to increase the female kids rate of dairy goats","volume":"201","author":"He","year":"2023","journal-title":"Theriogenology"},{"key":"ref101","doi-asserted-by":"publisher","first-page":"1117","DOI":"10.1093\/humrep\/dey081","article-title":"Creatine enhances the duration of sperm capacitation: a novel factor for improving in vitro fertilization with small numbers of sperm","volume":"33","author":"Umehara","year":"2018","journal-title":"Hum Reprod"},{"key":"ref102","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1016\/j.theriogenology.2024.10.015","article-title":"Expression of Tlr7\/8 in canine sperm and evaluation of the effect of ligand R848 on the sorting of canine X\/Y sperm","volume":"231","author":"Pan","year":"2025","journal-title":"Theriogenology"},{"key":"ref103","doi-asserted-by":"publisher","first-page":"104","DOI":"10.1016\/S0015-0282(16)45986-8","article-title":"Separation of human X and Y spermatozoa by albumin gradients and Sephadex chromatography","volume":"37","author":"Quinlivan","year":"1982","journal-title":"Fertil Steril"},{"key":"ref104","doi-asserted-by":"publisher","first-page":"678","DOI":"10.1016\/S0015-0282(16)49648-2","article-title":"Sex chromosome ratios determined by Karyotypic analysis in albumin-isolated human sperm","volume":"46","author":"Brandriff","year":"1986","journal-title":"Fertil Steril"},{"key":"ref105","first-page":"S88","article-title":"Determination of the ratio of X and Y bearing spermatozoa after albumin gradient method using double-labelled fluorescence in situ hybridization (Fish). Journal of the medical Association of Thailand =","volume":"79","author":"Aribarg","year":"1996","journal-title":"Chotmaihet Thangphaet"},{"key":"ref106","doi-asserted-by":"publisher","first-page":"661","DOI":"10.1016\/S0015-0282(16)47427-3","article-title":"Separation of human X- and Y-bearing sperm using Percoll density gradient centrifugation","volume":"40","author":"Kaneko","year":"1983","journal-title":"Fertil Steril"},{"key":"ref107","doi-asserted-by":"publisher","first-page":"565","DOI":"10.1023\/A:1022590321986","article-title":"Separating X-bearing human spermatozoa through a discontinuous Percoll density gradient proved to be inefficient by double-label fluorescent in situ hybridization","volume":"15","author":"Lin","year":"1998","journal-title":"J Assist Reprod Genet"},{"key":"ref108","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1080\/014850100262407","article-title":"Separation of sperm through a 12-layer Percoll column decreases the percentage of sperm staining with Quinacrine","volume":"44","author":"Check","year":"2000","journal-title":"Arch Androl"},{"key":"ref109","doi-asserted-by":"publisher","first-page":"e14226","DOI":"10.1111\/and.14226","article-title":"Identifying new sperm Western blot loading controls","volume":"53","author":"Feng","year":"2021","journal-title":"Andrologia"},{"key":"ref110","doi-asserted-by":"publisher","first-page":"235","DOI":"10.3390\/genes14010235","article-title":"Sorted Bulls\u2019 X-chromosome-bearing spermatozoa show increased Gapdhs activity correlating with motility","volume":"14","author":"Kapitonova","year":"2023","journal-title":"Genes"},{"key":"ref111","doi-asserted-by":"publisher","first-page":"1729","DOI":"10.5713\/ajas.18.0025","article-title":"Expression pattern of Prohibitin, capping actin protein of muscle Z-line Beta subunit and Tektin-2 gene in Murrah Buffalo sperm and its relationship with sperm motility","volume":"31","author":"Xiong","year":"2018","journal-title":"Asian Australas J Anim Sci"},{"key":"ref112","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1016\/j.cryobiol.2015.12.004","article-title":"Effect of cooling (4\u00b0C) and cryopreservation on cytoskeleton actin and protein tyrosine phosphorylation in Buffalo spermatozoa","volume":"72","author":"Naresh","year":"2016","journal-title":"Cryobiology"},{"key":"ref113","doi-asserted-by":"publisher","first-page":"46","DOI":"10.1016\/j.jri.2011.01.012","article-title":"Epitope selection to male specific antigens for sex selection in swine","volume":"89","author":"Mohammadi","year":"2011","journal-title":"J Reprod Immunol"},{"key":"ref114","doi-asserted-by":"publisher","first-page":"5994","DOI":"10.3390\/ijms24065994","article-title":"Nanobodies\u00ae: a review of diagnostic and therapeutic applications","volume":"24","author":"Jin","year":"2023","journal-title":"Int J Mol Sci"},{"key":"ref115","doi-asserted-by":"publisher","first-page":"466","DOI":"10.1111\/imr.13385","article-title":"Nanobody-based heavy chain antibodies and chimeric antibodies","volume":"328","author":"Koch-Nolte","year":"2024","journal-title":"Immunol Rev"}],"container-title":["Frontiers in Veterinary Science"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fvets.2025.1523491\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T07:56:01Z","timestamp":1741766161000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fvets.2025.1523491\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,12]]},"references-count":115,"alternative-id":["10.3389\/fvets.2025.1523491"],"URL":"https:\/\/doi.org\/10.3389\/fvets.2025.1523491","relation":{},"ISSN":["2297-1769"],"issn-type":[{"value":"2297-1769","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,12]]},"article-number":"1523491"}}