{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T02:30:56Z","timestamp":1790908256560,"version":"4.1.0"},"reference-count":33,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2019,11,14]],"date-time":"2019-11-14T00:00:00Z","timestamp":1573689600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Freemartinism in females born from heterosexual multiple pregnancies is characterized by the presence of XX\/XY cell lines due to the formation of a shared blood system by anastomoses between fetal membranes of co\u2013twins and leads to disturbed development of the reproductive system, including infertility. The aim of this study was to estimate the most precise and effective diagnostic method, especially useful for early identification of freemartinism in young female calves. The cytomolecular evaluation results of 24 Holstein-Friesian heifers from heterosexual twins was verified by molecular techniques: PCR, short tandem repeats (STRs), and relative quantitative PCR. The molecular analyses have been found to be a more efficient testing strategy, with a higher diagnostic success rate than karyotype evaluation. In 21 heifers, leucocyte chimerism determined by the 60, XX\/60, XY karyotype was revealed\u2014the proportion of the 60, XY male cell line in individual animals was in the range of 4\u201366%. In three cases, a normal karyotype 60, XX was identified, which indicates that anastomoses did not occur in 12.5% of studied twins and suggests that these potentially fertile heifers can be qualified for further breeding. The precise and early identification of freemartinism can be the basis for guidelines and selection recommendations concerning the reproductive performance of heifers born from heterosexual multiple pregnancies.<\/jats:p>","DOI":"10.3390\/ani9110971","type":"journal-article","created":{"date-parts":[[2019,11,14]],"date-time":"2019-11-14T10:56:34Z","timestamp":1573728994000},"page":"971","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Early Diagnostics of Freemartinism in Polish Holstein-Friesian Female Calves"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3706-4721","authenticated-orcid":false,"given":"Anna","family":"Kozubska-Soboci\u0144ska","sequence":"first","affiliation":[{"name":"Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5128-657X","authenticated-orcid":false,"given":"Grzegorz","family":"Smo\u0142ucha","sequence":"additional","affiliation":[{"name":"Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barbara","family":"Danielak-Czech","sequence":"additional","affiliation":[{"name":"Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,11,14]]},"reference":[{"key":"ref_1","first-page":"229","article-title":"New Aspects of Degenerative Changes in Reproductive System of Freemartin Heifers","volume":"11","author":"Rejduch","year":"2011","journal-title":"Ann. Anim. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"989","DOI":"10.1515\/aoas-2016-0028","article-title":"Cytogenetic and Molecular Diagnostics of XX\/XY Chimerism in Cattle, Sheep, and Goats\u2014A Review","volume":"16","author":"Rejduch","year":"2016","journal-title":"Ann. Anim. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"65","DOI":"10.22358\/jafs\/67389\/2004","article-title":"A Note on Cytogenetic Monitoring of Polish Red Cattle","volume":"13","author":"Rejduch","year":"2004","journal-title":"J. Anim. Feed Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.anireprosci.2004.09.008","article-title":"The Freemartin Syndrome: An Update","volume":"87","author":"Padula","year":"2005","journal-title":"Anim. Reprod. Sci."},{"key":"ref_5","unstructured":"Esteves, A., B\u00e5ge, R., and Payan Carreira, R. (2012). Freemartinism in Cattle. Ruminants: Anatomy, Behavior and Diseases, Nova Science Publishers Inc."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e4616","DOI":"10.7717\/peerj.4616","article-title":"Applying Real-Time Quantitative PCR to Diagnosis of Freemartin in Holstein Cattle by Quantifying SRY Gene: A Comparison Experiment","volume":"6","author":"Qiu","year":"2018","journal-title":"PeerJ"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1111\/j.1365-2052.2010.02030.x","article-title":"Validation of Whole Genome Linkage-Linkage Disequilibrium and Association Results, and Identification of Markers to Predict Genetic Merit for Twinning","volume":"41","author":"Bierman","year":"2010","journal-title":"Anim. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"445","DOI":"10.17221\/7707-CJAS","article-title":"A High Incidence of Leukocyte Chimerism (60, XX\/60, XY) in Single Born Heifers Culled Due to Underdevelopment of Internal Reproductive Tracts","volume":"59","author":"Szczerbal","year":"2014","journal-title":"Czech J. Anim. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"239","DOI":"10.17221\/4306-CJAS","article-title":"The Ambiguity of Freemartinism Diagnosis in Cattle Revealed by Cytogenetic and Molecular Techniques","volume":"49","author":"Switonski","year":"2004","journal-title":"Czech J. Anim. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1262\/jrd.2012-070","article-title":"Evidence for Almost Complete Sex-Reversal in Bovine Freemartin Gonads: Formation of Seminiferous Tubule-like Structures and Transdifferentiation into Typical Testicular Cell Types","volume":"58","author":"Harikae","year":"2012","journal-title":"J. Reprod. Dev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1051\/rnd:19880709","article-title":"Anti-M\u00fcllerian Hormone and Freemartinism: Inhibition of Germ Cell Development and Induction of Seminiferous Cord-like Structures in Rat Fetal Ovaries Exposed in Vitro to Purified Bovine AMH","volume":"28","author":"Vigier","year":"1988","journal-title":"Reprod. Nutr. Dev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"R21","DOI":"10.1530\/JME-14-0018","article-title":"Molecular Mechanisms Involved in Mammalian Primary Sex Determination","volume":"53","author":"She","year":"2014","journal-title":"J. Mol. Endocrinol."},{"key":"ref_13","first-page":"55","article-title":"Genetic Aspects of Twinning in Cattle","volume":"43","author":"Komisarek","year":"2002","journal-title":"J. Appl. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/S0021-9975(08)80267-8","article-title":"Retrospective Studies on the Measurements, Karyotyping and Pathology of Reproductive Organs of Bovine Freemartins","volume":"110","author":"Khan","year":"1994","journal-title":"J. Comp. Pathol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1672","DOI":"10.2460\/javma.1994.204.10.1672","article-title":"Diagnosis of Freemartinism in Cattle: The Need for Clinical and Cytogenic Evaluation","volume":"204","author":"Zhang","year":"1994","journal-title":"J. Am. Vet. Med. Assoc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1159\/000117716","article-title":"XX\/XY Chimerism in Cattle: Clinical and Cytogenetic Studies","volume":"2","author":"Peretti","year":"2008","journal-title":"Sex. Dev."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1159\/000245911","article-title":"Classical and Molecular Cytogenetics of Disorders of Sex Development in Domestic Animals","volume":"126","author":"Parma","year":"2009","journal-title":"Cytogenet. Genome Res."},{"key":"ref_18","first-page":"308","article-title":"Preliminary Identification of the Fragile X in Two Crossbred Cows","volume":"45","author":"Slota","year":"2000","journal-title":"Vet. Med. (Praha)"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1111\/j.1439-0388.2004.00456.x","article-title":"The Application of Genetic Markers for Cell Chimerism Diagnosis in Lambs","volume":"121","author":"Rejduch","year":"2004","journal-title":"J. Anim. Breed. Genet."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1159\/000245905","article-title":"Fluorescence in Situ Hybridization Applied to Domestic Animal Cytogenetics","volume":"126","author":"Rubes","year":"2009","journal-title":"Cytogenet. Genome Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"311","DOI":"10.17221\/5628-VETMED","article-title":"The Phenomenon of Cell Chimerism in Goats","volume":"50","author":"Rychlik","year":"2012","journal-title":"Veterin\u00e1rn\u00ed Medic\u00edna"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1016\/j.theriogenology.2007.06.022","article-title":"Diagnosis of Bovine Freemartinism by Fluorescence in Situ Hybridization on Interphase Nuclei Using a Bovine Y Chromosome-Specific DNA Probe","volume":"68","author":"Sohn","year":"2007","journal-title":"Theriogenology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1116","DOI":"10.1016\/j.theriogenology.2014.01.040","article-title":"The Use of a Novel Combination of Diagnostic Molecular and Cytogenetic Approaches in Horses with Sexual Karyotype Abnormalities: A Rare Case with an Abnormal Cellular Chimerism","volume":"81","author":"Anaya","year":"2014","journal-title":"Theriogenology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1177\/104063870601800508","article-title":"Comparison of Cytogenetics and Polymerase Chain Reaction Based Detection of the Amelogenin Gene Polymorphism for the Diagnosis of Freemartinism in Cattle","volume":"18","author":"McNiel","year":"2006","journal-title":"J. Vet. Diagn. Investig. Off. Publ. Am. Assoc. Vet. Lab. Diagn. Inc."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1148","DOI":"10.1016\/j.theriogenology.2009.06.029","article-title":"Freemartinism and FecXR Allele Determination in Replacement Ewes of the Rasa Aragonesa Sheep Breed by Duplex PCR","volume":"72","author":"Dervishi","year":"2009","journal-title":"Theriogenology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1111\/j.1365-2052.2010.02099.x","article-title":"Chimaerism Detection in Bovine Twins, Triplets and Quadruplets Using Sex Chromosome-Linked Markers","volume":"42","author":"Ron","year":"2011","journal-title":"Anim. Genet."},{"key":"ref_27","first-page":"175","article-title":"Application of Hormonal and Single Multiplex PCR Assays for Detection of Freemartinism in a Horned Goat","volume":"38","author":"Pourjafar","year":"2012","journal-title":"J. Fac. Vet. Med. Istanb. Univ."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.jbbm.2004.05.004","article-title":"Refined Quantitative Fluorescent PCR of Y-Chromosome DNA Sequences Mosaics in Turner\u2019s Syndrome Patients\u2014Alternative to Real-Time PCR","volume":"60","author":"Vodicka","year":"2004","journal-title":"J. Biochem. Biophys. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1002\/pd.2424","article-title":"Combined QF-PCR and MLPA Molecular Analysis of Miscarriage Products: An Efficient and Robust Alternative to Karyotype Analysis","volume":"30","author":"Donaghue","year":"2010","journal-title":"Prenat. Diagn."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"6379","DOI":"10.4238\/2013.December.9.1","article-title":"Validation of Quantitative Fluorescent-PCR for Rapid Prenatal Diagnosis of Common Aneuploidies in the Chinese Population","volume":"12","author":"Xu","year":"2013","journal-title":"Genet. Mol. Res. GMR"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4618","DOI":"10.1182\/blood.V99.12.4618","article-title":"Quantitative Assessment of Hematopoietic Chimerism after Bone Marrow Transplantation by Real-Time Quantitative Polymerase Chain Reaction","volume":"99","author":"Alizadeh","year":"2002","journal-title":"Blood"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1090","DOI":"10.1111\/j.1464-5491.2009.02846.x","article-title":"Genome-Wide SNP Genotyping Study Using Pooled DNA to Identify Candidate Markers Mediating Susceptibility to End-Stage Renal Disease Attributed to Type 1 Diabetes","volume":"26","author":"Craig","year":"2009","journal-title":"Diabet. Med."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"279","DOI":"10.2478\/aoas-2014-0011","article-title":"The Effect of Using DNA Obtained from Blood of Cattle with Genetic Chimerism on Illumina\u2019s Beadchip Assay Performance","volume":"14","author":"Gurgul","year":"2014","journal-title":"Ann. Anim. Sci."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/9\/11\/971\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:34:25Z","timestamp":1760189665000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/9\/11\/971"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,11,14]]},"references-count":33,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2019,11]]}},"alternative-id":["ani9110971"],"URL":"https:\/\/doi.org\/10.3390\/ani9110971","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,11,14]]}}}