Cryoinjuries in Cryopreserved Semen of Ichthyoelephas longirostris with Ethylene Glycol

<i>Ichthyoelephas longirostris</i>, commonly known as pataló, is an endemic species from Colombia, categorized as endangered due to habitat degradation and overfishing. In this context, semen cryopreservation emerges as an effective strategy for its conservation. This study evaluated the damage caused by semen cryopreservation using a cryosolution composed of ethylene glycol (EG) at three inclusion levels (6%, 8%, and 10% <i>v</i>/<i>v</i>), skimmed milk powder (3% <i>w</i>/<i>v</i>), and glucose (6% <i>w</i>/<i>v</i>), all prepared in distilled water. A randomized experimental design was employed, with three treatments and a control (fresh semen), using pooled semen from different males (<i>n</i> = 12). Each sample was analyzed in triplicate. Sperm parameters were evaluated through computer-assisted analysis (CASA), and damage to the plasma membrane, mitochondria, and DNA was assessed via flow cytometry. Fresh semen showed low levels of membrane damage (9.6 ± 6.9%), mitochondrial damage (29.1 ± 16.8%), and DNA fragmentation (0.24 ± 0.13%). In thawed semen, the highest membrane damage was observed with 10% EG (79.3 ± 13.0%), while the highest DNA fragmentation occurred with 6% EG (22.5 ± 21.9%). The cryosolution composed of 8% EG showed the best results, with lower levels of damage in the evaluated structures. These findings suggest that the combination of 8% EG, 3% skimmed milk powder, and 6% glucose is an effective formulation to reduce cellular damage during <i>I. longirostris</i> semen cryopreservation, representing a useful tool for conservation programs of this species.