{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T10:09:04Z","timestamp":1789380544159,"version":"build-2803163510"},"reference-count":34,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Talent Introduction Program of \u201cTianchi Talents\u201d"},{"name":"Modern Agricultural Sheep Industry Technology System of the Autonomous Region","award":["XJARS-09-202608"],"award-info":[{"award-number":["XJARS-09-202608"]}]},{"name":"Major Special Project of the Autonomous Region","award":["2023A02011-2"],"award-info":[{"award-number":["2023A02011-2"]}]},{"name":"Scientific and Technological Innovation Team for Precise Breeding Technology of Cattle and Sheep","award":["2024TSYCTD0012"],"award-info":[{"award-number":["2024TSYCTD0012"]}]},{"name":"Tianshan Talents Program of Autonomous Region","award":["2023TSYCJC0031"],"award-info":[{"award-number":["2023TSYCJC0031"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>This study aimed to explore whether zona-associated residual sperm may contribute to exogenous Y-DNAY-DNA contamination and cause sex bias in PCR-based sex classification of ovine early-stage embryos, based on the amplification of the AMEL gene. Primers targeting the conserved regions of the ovine AMELX and AMELY genes were designed, and a nested PCR system using AMEL and YWHAZ genes was established. Group A comprised 17 female and 33 male embryos, corresponding to a female-to-male ratio of 0.52:1, whereas Group B comprised 23 female and 27 male embryos, corresponding to a female-to-male ratio of 0.85:1. DAPI staining revealed sperm-morphology structures or structures containing sperm-derived DNA adjacent to the zona pellucida of ovine early-stage embryos. Sequence alignment of PCR amplicons showed consistency with the AMELX and AMELY reference genes. In conclusion, these results support our research hypothesis that zona-associated residual sperm may serve as a potential source of Y-DNAY-DNA contamination and interfere with PCR-based embryonic sex determination.<\/jats:p>","DOI":"10.3390\/ani16182888","type":"journal-article","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T09:34:01Z","timestamp":1789378441000},"page":"2888","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Analysis of Zona-Associated Sperm as a Potential Source of Exogenous Y-DNA Interference in Sex-Typing of In Vitro-Fertilized Ovine Embryos"],"prefix":"10.3390","volume":"16","author":[{"given":"Qiangqiang","family":"Ma","sequence":"first","affiliation":[{"name":"College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China"},{"name":"Key Laboratory of Genetic Breeding and Reproduction of Herbivorous Livestock, Ministry of Agriculture and Rural Affairs\/Xinjiang Key Laboratory of Reproductive Regulation and Breeding for Ruminant Livestock\/Xinjiang Key Laboratory of Animal Biotechnology, Xinjiang Academy of Animal Science, Urumqi 830011, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"conceptualization","vocabulary":"credit"}]},{"given":"Ying","family":"Chen","sequence":"additional","affiliation":[{"name":"Key Laboratory of Genetic Breeding and Reproduction of Herbivorous Livestock, Ministry of Agriculture and Rural Affairs\/Xinjiang Key Laboratory of Reproductive Regulation and 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