Pharmacologically assisted semen collection and sperm morphology assessment methods in wild six-banded armadillos (Euphractus sexcinctus)

Six-banded armadillos (Euphractus sexcinctus), a non-endangered species, serve as a valuable experimental model for developing assisted reproductive technologies aimed at conserving endangered species within the order Cingulata. However, limited knowledge of their reproductive biology presents a critical challenge. This study aimed to evaluate the effectiveness of a pharmacological semen collection protocol and compare Karras-modified staining using brightfield microscopy (KA), phase contrast microscopy (PC), and differential interference contrast microscopy (DIC) for assessing sperm morphology. Twenty free-ranging male six-banded armadillos were captured and subjected to the following pharmacological sedation protocol: butorphanol tartrate, detomidine hydrochloride and midazolam hydrochloride (i.m., 0.1 mg/kg each). Ten minutes after administration, semen was extracted by applying gentle pressure along the penis, from its base to the tip. The expelled semen, which accumulated into the urethral fossa, was collected using a variable-volume micropipette and assessed for macro and microscopic characteristics. Ninety-five percent of the males exhibited a partial ejaculation, with no aversive behavior or signs of discomfort observed. Most semen samples (83 %) contained more than 30 % of morphologically abnormal sperm with considerable individual variability in sperm defect profiles. While all microscopy techniques allowed effective assessment of sperm morphology, KA was the most efficient in identifying acrosome integrity and head abnormalities. This method offers a practical and cost-effective solution, particularly suited for field conditions. This study reports the first successful pharmacological protocol for semen collection in six-banded armadillos, highlighting its potential use for reproductive assessments and contributing to developing conservation strategies for related species.