The cryopreservation of sperm from different segments of the epididymis is the ultimate opportunity to preserve the gene pool of animals of great breeding value. Despite the fact that samples from the caput epididymis are immotile, their DNA integrity could justify their use in ICSI procedures. Thus, the objective of the present study was to evaluate the DNA integrity and sperm motility of stallion sperm collected from different regions of the epididymis. Equine epididymides were obtained from 10 stallions using retrograde flush with 40mL of Botu-Semen per caudae epididymis. Sperm from the caudae epididymis were obtained with a slicing technique in association with a float-up method. The samples were divided into 3 groups: Botu-Semen (control), Fert-Talp and Sperm-Talp, and diluted 1:1 with these media. The samples were centrifuged (1000 x g/10 min) to concentrate the sperm, the supernatant was removed, and the pellets were resuspended in Botu-Crio . Samples were loaded into 0.5mL straws and cooled at 5 C for 20 min. They were then frozen in nitrogen vapor (6 cm above the level of liquid nitrogen) for an additional 20 min, plunged into liquid nitrogen and stored. Sperm chromatin structure assay was performed using a semen aliquot diluted in 200 mL of buffer solution (0.186g disodium EDTA, 0.790 g Tris-HC1, 4.380 g NaC1 in 500 mL deionized water, pH 7.4). This was added with 400 mL of detergent/acid solution. Thirty seconds later, 1.2 mL acridine orange solution was added (Evenson, In: Sorsa M, Norppa H (ed), Monitoring of