Simple SummaryThe collared peccary is a wild ungulate important for the maintenance of ecosystems in the Americas. The in vitro fertilization (IVF) technique can contribute to the conservation of and knowledge about the reproductive characteristics of this species. The collared peccary’s spermatozoa are sensitive to stress conditions. Therefore, the steps of seminal plasma (SP) removal and sperm selection for fertilization need to be determined. Consequently, the most used methods in domestic ungulate species, namely, swim-up, Percoll®gradient (PG), and washing by centrifugation (WC), were evaluated. Initially, it was observed that regardless of the sample preparation for the swim-up (with SP or without SP), this method was not efficient in isolating motile sperm. Different PG concentrations were compared, revealing PG 45–90% recovered spermatozoa with better motility patterns compared to PG 35–70% and non-selected sperm. Then, PG 45–90% was compared to WC for sperm motility and heterologous IVF with swine oocytes. PG 45–90% showed better motility patterns, but the rate of fertilization and development of embryos were similar compared to WC. Therefore, PG 45–90% and WC are recommended for SP removal and sperm selection in the collared peccary.AbstractMethods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll®gradient densities (PG 45–90% and PG 35–70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45–90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35–70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45–90% was used for fertilization. PG 45–90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2–26.3%) and morula (8.1–10.5%) rates did not differ between the groups. Therefore, PG 45–90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.