A B S T R A C T The conservation of semen may have its costs reduced when used within a certain period of time through cooling techniques. These techniques are alternatives to reproductive management in fish farms, optimizing fertilization of oocytes and ensuring satisfactory reproduction. The quality of the extenders is essential, since it guarantees the maintenance of sperm cells. The tambaqui (Colossoma macropomum) is an endemic species from Brazil and reared in tropical regions throughout the world. Its supply chain is trending upward and production technologies are being improved, both by governmental programs and private initiative, as is the case of fish farms such as Delicious Fish in the state of Mato Grosso, Brazil, where this study was carried out. Different concentrations of cryoprotectants added in the extenders were tested during cooling of fresh semen of C. macropomum, and their motility was analyzed. Six males with secondary reproductive attributes of Neotropical migratory species were selected, weighed and hormonally induced. After stripping the semen through anteroposterior massage, a pool of semen from all the animals was constituted. The extenders tested were: T1: BTS (Beltsville Thawing Solution); T2: BTS + 1% Me2SO (dimethyl sulfoxide); T3: BTS + 2.5% Me2SO; T4: BTS + 5% Me2SO; T5: BTS + 8% Me2SO; T6: BTS + 1% Me2NCHO (dimethylformamide); T7: BTS + 2.5% Me2NCHO; T8: BTS + 5% Me2NCHO; T9: BTS + 8% Me2NCHO e T10: cooled pure semen. The progressive motility (PM%) of the samples was analyzed. After the analyses, the pool of semen was kept in eppendorfs, one for each analysis per day, and stored in refrigeration at 4 ± 1 °C. The seminal parameters were evaluated every 24 h to observe sperm viability. The statistic analyses were performed using the STATISTICA software. The motility of the semen “in natura” for each treatment was 95%, 88%, 78%, 85%, 67%, 72%, 60%, 57%, 47%, 83%, for the treatments T1, T2, T3, T4, T5, T6, T7, T8, T9 and T10, respectively. The progressive motility rate gradually decreased over time, varying according to the extenders. After four days the motility was 32% (T1), 33% (T2), 37% (T3), 27% (T4), 12% (T5), 38% (T6), 30% (T7), 22% (T8), 8% (T9) e 12% (T10). The increase in concentration of both internal cryoprotectants showed no significant difference for the treatments, except for T9, at 5% of significance (p < 0.05). The use of BTS without internal cryoprotectant did not reduce the efficiency of preservation of semen. However, the increase in the concentration of internal cryoprotectant has demonstrated a reduced percentage rate, particularly at concentrations of 8%. According to the extenders used, BTS + 1% of Me2NCHO presented the best sperm motility.