In asses, semen collection, cryopreservation, and artificial insemination (AI) with frozen-thawed semen have been scarcely described and success rate, particularly following AI, is reportedly low. In the absence of reliable protocols, assisted reproductive technologies cannot support the conservation efforts aimed at endangered wild ass species and domestic donkey breeds. Two experiments were conducted in this study. In experiment 1 we evaluated freezing Abyssinian donkey (N = 5, 4 ejaculates each) spermatozoa using three freezing extenders (Berliner Cryomedium + glycerol, BC+G; BotuCrio, BOTU; INRAFreeze, INRA) and two cryopreservation techniques (liquid nitrogen vapour, LNV; directional freezing, DF). Post-thaw evaluation indicated that BOTU and INRA were similar and both superior to BC+G (P ≤ 0.004 for all motility tests), and that DF was superior to LNV (P < 0.002 for all evaluation parameters). In experiment 2, relying on these results, we used Abyssinian donkey sperm frozen in BOTU and INRA by DF for AI (N = 20). Prior to AI, thawed samples were diluted in corresponding centrifugation media or autologous seminal fluids at 1:1 ratio. No difference was found between BOTU and INRA or between the addition of seminal fluids or media, all resulting in ~50% pregnancy, and no differences were noted between males (N = 4). The size of pre-ovulatory follicle was a significant (P = 0.001) predictor for AI success with 9/10 pregnancies occurring when follicular size ranged between 33.1-37.4 mm, no pregnancy when it was smaller, and only one when larger. A number of ass species face the risk of extinction. Knowledge gained in this study on the Abyssinian donkey can be customised and transferred to its closely related endangered species and breeds.