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The donkey jack sex glands are larger than stallions, responsible for producing most part of seminal plasma and the second fraction of ejaculate, along with epididymis tail. Plugged ampullae occur by sperm accumulation obstructing the lumen, inducing decrease in sperm quality and may cause azoospermia. In this study, a Pêga breed donkey jack, aging 4 years, was evaluated for breeding soundness evaluation due to a sudden decrease in semen parameters and low fertility rates. Palpation, measurements, and ultrasound examinations of testicles were normal; however, rectal palpation revealed increased volume of ampullae and deferent duct, and the transrectal ultrasonography revealed distended ampullae with hyperechogenic material in the ampullae lumen. After ampullae massage, the semen was collected with artificial vagina for evaluation, resulting in high concentrated semen (1.46 × 109 spermatozoa/mL) with low motility (5%), 14% of major defects, and 57% of minor defects. Plugged ampullae were suggested, and the treatment was performed by ampullae massage per rectum and three consecutive semen collections associated with the parenteral use of oxytocin 20 IU iv, aiming to discharge the semen accumulation. Daily regimen of semen collection was recommended during 10 days, and after this time, semen was collected at least three times a week. The semen parameters restored to normal (80% motility) after 30 days. The donkey jack returned to the breeding season with a regimen of 3 days a week of semen collection.Donkeys have large accessory sex gland than stallion.Plugged ampullae occur by sperm accumulation obstructing the lumen.This is the first report of plugged ampullae in donkey stallion.Treatment of ampullary spermastasis in donkey is similar to reported in stallion.