Generation of Biparental Bovine Embryos via Haploid Cell Injection (Semi-cloning) for Embryo Production with Predetermined Parental Genomes.

This protocol describes a method for generating biparental bovine embryos with predetermined paternal genomes using haploid androgenetic blastomere injection (semi-cloning). The approach enables replacement of sperm at fertilization with pre-genotyped haploid embryonic cells, allowing controlled paternal genome selection prior to embryo reconstruction. The protocol involves producing androgenetic haploid embryos via oocyte enucleation at telophase II, following intracytoplasmic sperm injection (ICSI)-mediated activation. Haploid androgenetic blastomeres are isolated from morula-stage embryos, prepared for micromanipulation, and injected into mature oocytes using a piezo-assisted intracytoplasmic injection system. Reconstructed embryos are subsequently cultured in vitro and may be transferred to recipient females to obtain gestations and offspring with the chosen paternal genome. This method integrates established reproductive technologies, including oocyte selection, in vitro maturation, intracytoplasmic sperm injection, telophase II enucleation, and piezo-assisted cell microinjection, into a unified workflow adapted for bovine embryo production. The protocol provides a platform for sperm genome amplification, genomic characterization of haploid blastomeres prior to use, and reconstruction of embryos with defined paternal genetic composition. Semi-cloned embryos produced using this approach develop to the blastocyst stage in vitro and can establish pregnancies following embryo transfer. This protocol is applicable to researchers in cattle reproductive biotechnology, livestock genetics, and developmental biology, and supports applications in genomic selection, functional genomics, and genetic conservation. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Micropipette production Basic Protocol 2: Micromanipulator setup Basic Protocol 3: Preparation of micromanipulation dishes Basic Protocol 4: Intracytoplasmic sperm injection Basic Protocol 5: Zygote enucleation Basic Protocol 6: Intracytoplasmic haploid cell injection (ICHI).