Evaluation of Proliferative Potential and Collagen Deposition in Vitrified Canine Testicular Fragments Subjected to Different Cryoprotectant Combinations and Warming Temperatures

Testicular cryopreservation is a promising tool for fertility preservation, with applications in endangered species, prepubertal animals and individuals of high genetic value. This study evaluated proliferative potential and collagen deposition in adult canine testicular fragments subjected to vitrification using different warming temperatures (37°C for 1 min and 50°C for 5 s) and cryoprotectant combinations: ethylene glycol/glycerol (EG/GLY), dimethyl sulfoxide/glycerol (DMSO/GLY) and dimethyl sulfoxide/ethylene glycol (DMSO/EG). Twenty‐four testicular fragments (3 × 1 × 1 mm) per dog were collected from 10 adult dogs, vitrified using the needle immersion method and subsequently warmed according to the experimental protocol. Proliferative potential was analysed using AgNOR staining, and collagen deposition was assessed using Picrosirius staining. AgNOR analysis indicated that proliferative potential was maintained across all groups, with no significant differences ( p > 0.05), ranging from 2.42 ± 0.05 (EG/GLY37) to 2.71 ± 0.31 (DMSO/EG50). Picrosirius staining revealed increased collagen only in the DMSO/GLY37 group (194.80 ± 17.14, p < 0.05), with a significant increase in type III collagen fibres ( p < 0.05). All other groups maintained collagen levels similar to those of the control group (171.20 ± 18.80, p > 0.05). In conclusion, the tested cryoprotectant combinations and warming temperatures adequately preserved proliferative potential, and collagen organization in vitrified adult canine testicular fragments, except DMSO/GLY37, supporting their application in assisted reproductive technologies & species conservation programmes.