Ruiz-Díaz, SaraMaroto, MaríaFernández-Gonzalez, Raúlde Frutos, CeliaLombó, MartaArias Álvarez, María2025-07-042025-07-042025Ruiz-Díaz, S., Maroto, M., Fernández-Gonzalez, R., de Frutos, C., Lombó, M., & Arias-Álvarez, M. (2025). Effect of 16h of rabbit sperm incubation on capacitation and heterologous in vitro fertilization. Animal reproduction science, 277, 107841. https://doi.org/10.1016/j.anireprosci.2025.1078410378-432010.1016/j.anireprosci.2025.107841https://hdl.handle.net/20.500.14352/122216Justificación de autores: Fernandez Gonzalez Raul: Writing – review & editing, Methodology, Investigation. Maroto Maria: Writing – review & editing, Methodology, Investigation. Arias Maria: Writing – review & editing, Writing – original draft, Funding acquisition, Formal analysis, Conceptualization. Lombó Marta: Writing – review & editing, Methodology, Investigation. de Frutos Celia: Writing – review & editing, Methodology, Investigation. Ruiz Diaz Sara: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Becas: FJC2019-040385-I IND2018/BIO-9610In vitro fertilization (IVF) in rabbits is an important biotechnology, but its success remains low due to inefficient sperm capacitation. This study examines relationships between motility parameters (MP), protein tyrosine phosphorylation (PTP) and acrosomal exocytosis (AE) between sperm recovered after direct swim-up (DSU), and after 16 h of in vitro incubation under capacitating conditions, and heterologous IVF capacity. Variables were assessed in fresh ejaculates (ES), and after 2 h of DSU, and 16 h (T16) of incubation. PTP were grouped into four patterns in the different sperm structures according to their time-dependent appearance and AE. Throughout incubation, MP remained stable whereas AE increased after DSU and 16 h of incubation. Mid-piece PTP patterns increased after DSU and persisted until 16 h in reacted (R) and non-reacted spermatozoa (NR). In R and NR, annulus-ring PTP levels were also significantly higher in T16 versus DSU or ES. Flagellum PTP levels rose significantly during incubation such that highest levels were recorded in T16. In R sperm, levels of PTP patterns associated with the equatorial zone in T16 were significantly higher compared to NR and to DSU and ES. Cleavage rates were significantly higher at T16 than DSU and these were similar to rates recorded for homologous IVF used as positive control. Our capacitation protocol and PTP patterns at 16 h rendered the sperm competent to fertilize an oocyte in vitro. This time point matches the physiological timing of fertilization in vivo. We also describe a new annulus ring PTP pattern for this species.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/journal article1873-2232https://doi.org/10.1016/j.anireprosci.2025.10784140306182open access636.082.4Sperm capacitationAcrosome reactionProtein tyrosine phosphorylationAnnulus-ringAssisted reproductive technologiesRabbitProducción animal3104 Producción Animal