CONTEXT: C-type natriuretic peptide (CNP)-based pre-maturation can improve bovine oocyte developmental competence but adds a separate culture and transfer step to in vitro embryo production (IVP). AIMS: To develop a one-step CNP and 17β-estradiol (E2) modification of conventional in vitro maturation (IVM), demonstrate its application, and assess timed amphiregulin (AREG) supplementation. METHODS: Two experiments used 3182 abattoir-derived cumulus-oocyte complexes. Experiment 1 compared conventional 24-h IVM (control), CNP during recovery only (recovery-only), 6-h CNP-E2 pre-IVM followed by 24-h IVM (biphasic), and 24- or 30-h CNP-E2 IVM (one-step 24-h and one-step 30-h). Experiment 2 compared control, one-step 24-h, and one-step 24-h + AREG, with AREG added after 6 h. Cleavage and blastocyst development were assessed on Days 3 and 7-8, respectively. KEY RESULTS: In Experiment 1, biphasic and one-step 24-h increased Day 8 total blastocyst yield relative to control (26.5% vs 19.7%, P = 0.022; and 25.7% vs 19.7%, P = 0.042, respectively). In Experiment 2, one-step 24-h increased Day 7 total blastocyst yield (31.6% vs 23.8%; P = 0.017). One-step 24-h + AREG increased Day 8 hatching blastocysts relative to control (16.0% vs 9.9%; P = 0.013) but not relative to one-step 24-h. CONCLUSIONS: Direct CNP-E2 supplementation during 24-h IVM eliminated the separate pre-IVM transfer while improving blastocyst endpoints relative to conventional IVM. IMPLICATIONS: The modification preserves a conventional 24-h IVM schedule; however, external validation using embryo-quality and post-transfer outcomes is warranted.