Nile tilapia Oreochromis niloticus is a popular freshwater fish that has been extensively and intensively cultured worldwide. However, cryopreservation of its sperm, especially pre-freezing procedure, has not been properly developed. Therefore, the study aimed to determine the best pre-freezing procedure for cryopreservation of Nile tilapia Oreochromis niloticus sperm. The completely randomized design with five treatments and four replications was employed in this study. The tested treatments were T1=4°C → 0°C → -4°C → -10°C → -79°C → -196°C, T2=4°C → 0°C → -4°C → -10°C → -196°C, T3=4°C → 0°C → -4°C → -196°C, T4=4°C → 0°C → -196°C, and T5=4°C → -196°C, with a 10 min equilibration at each respective temperature. Furthermore, sperm was cryopreserved for two weeks in liquid nitrogen (-179°C). The results of the ANOVA test showed that pre-freezing had a significant effect on sperm motility, and viability (P<0.05), but had no considerable impact on sperm abnormality (P>0.05). Treatment T4 exhibited higher motility and viability, but these values were not significantly different from T3 and T5. Based on practical consideration, it was recommended to utilize the T5 pre-freezing procedures (4°C → -196°C) for cryopreservation of Nile tilapia sperm. Considering these results, Nile tilapia sperm can be directly cryopreserved into liquid nitrogen after equilibration at 4°C for 10 min.
