Comparative Evaluation of Staining Techniques in Thawed Cryopreserved Dog Semen

(1) Background: Accurate assessment of sperm morphology is essential for evaluating the quality of cryopreserved canine semen used in artificial insemination and for improving cryopreservation protocols. This study compared six staining techniques, Eosin, Eosin–Nigrosin, Diff-Quick^®, Hemacolor^®, Spermac^®, and Formol-citrate Bengal Rose, for light-microscopic evaluation of frozen–thawed canine spermatozoa. (2) Methods: Semen from ten dogs was thawed, divided into four aliquots, and either left untreated or exposed to thermal stress at 6 °C, 18 °C, or 37 °C for two hours to induce morphological variation. A total of 360 slides and 960 evaluations were performed immediately after staining and again after 24 h, 7 days, and 3 months to assess staining quality and stability over time. (3) Results: Eosin produced stable staining for up to three months and was the most economical method, though its initial detail recognition was lower than that of Spermac^® and Formol-citrate Bengal Rose. Eosin–Nigrosin showed reduced contrast and detail. Diff-Quick^® provided better contrast than Eosin–Nigrosin, while Hemacolor^® maintained consistent quality regardless of stress treatment or storage duration. Spermac^® yielded the highest initial morphological detail but deteriorated during storage. Formol-citrate Bengal Rose combined high detail recognition with stable staining throughout the study. Cryopreservation increased looped tails, and incubation at 37 °C markedly elevated pathological sperm and rudimentary tails. (4) Conclusions: All six staining methods were suitable for evaluating and archiving frozen–thawed canine semen. Formol-citrate Bengal Rose and Spermac^® offered the best detail, while Eosin provided a cost-effective option with excellent long-term stability.