{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T04:54:53Z","timestamp":1790916893062,"version":"4.1.0"},"reference-count":65,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Key Research and Development Program of Ningxia Hui Autonomous Region","award":["2024BBF01005"],"award-info":[{"award-number":["2024BBF01005"]}]},{"award":["2024BBF01005"],"award-info":[{"award-number":["2024BBF01005"]}],"id":[{"id":"https:\/\/ror.org\/015bdn754","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Shaanxi Provincial Science and Technology Plan Project\u2014Key Project\u2014Core Key Technology Tackling\u2014Agricultural and Rural Areas","award":["2024NC2-GJHX-13"],"award-info":[{"award-number":["2024NC2-GJHX-13"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Sex-sorted sperm permit the preferential production of offspring of a specified sex, but flow sorting and cryopreservation can compromise sperm function and reduce the efficiency of conventional in vitro fertilization (IVF). We sequentially evaluated oocyte maturation duration, sperm processing, post-injection incubation medium, and nine activation regimens using slaughterhouse-derived oocytes, and then evaluated the best-performing combination under the tested sequential conditions using ovum pick-up (OPU)-derived oocytes. In conventional IVF, the presumptive normal fertilization rate, based on the presence of two pronuclei (2PN), was 2.45% with X-sorted sperm and 46.13% with conventional sperm. The final selected combination, consisting of 22 h maturation, Brackett\u2013Oliphant (BO)-based swim-up, oocyte maturation medium (OM) during post-injection incubation, and treatment with 7% ethanol for 5 min [ETH (5)], resulted in a presumptive normal fertilization (2PN) rate of 52.92%, a blastocyst rate of 10.46%, and an ICM\/TE ratio of 0.82. In the validation using OPU-derived oocytes, the blastocyst formation rate after ssICSI was 13.46% when calculated relative to injected oocytes and 16.84% when calculated relative to surviving oocytes. These rates differed significantly from the blastocyst formation rate after ssIVF (7.88%), whereas cleavage rates did not differ significantly between groups. ETH (5) provided the most favorable in vitro balance of presumptive 2PN formation, blastocyst development, and ICM\/TE cell allocation among the tested regimens. Because the factors were optimized sequentially, their interactions were not estimated, which represents a limitation of this study. Furthermore, implantation, pregnancy, and calving rates require validation in independent in vivo embryo-transfer experiments.<\/jats:p>","DOI":"10.3390\/ani16192983","type":"journal-article","created":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T14:45:10Z","timestamp":1790865910000},"page":"2983","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimization of a Bovine Intracytoplasmic Sperm Injection System Using Sex-Sorted Sperm: Screening with Slaughterhouse-Derived Oocytes and Validation with Ovum Pick-Up-Derived Oocytes"],"prefix":"10.3390","volume":"16","author":[{"given":"Xuexiao","family":"Song","sequence":"first","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"conceptualization","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Tao","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"}]},{"given":"Hongxing","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"}]},{"given":"Heqiang","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Baobao","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"}]},{"given":"Yanfang","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Zheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"}]},{"given":"Dexiang","family":"Fan","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Yaochang","family":"Wei","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Dong","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"supervision","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Yongsheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Xianyang 712100, China"},{"name":"Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Xianyang 712100, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Unnikrishnan, V., Kastelic, J., and Thundathil, J. 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