Cryopreservation of testicular tissue from pubertal dogs (Canis lupus familiaris) comparing slow freezing and solid surface vitrification methods.

Cryopreservation of testicular tissue is crucial for germplasm banking in canine breeding programs, conservation of endangered canids, and veterinary oncology. However, optimal methods for canine testicular tissue remain underexplored due to challenges like ice crystal formation in slow freezing and cryoprotectant (CPA) toxicity in vitrification. This study compared automated slow freezing (ASF) and solid surface vitrification (SSV) for preserving histo-architecture and cellular function in testicular tissue from pubertal dogs. Testes from 15 pubertal dogs were fragmented into 5×5×5 mm pieces, distributed into control (CG), SSV, and ASF groups, cryopreserved, stored in liquid nitrogen for 15 days, thawed, and analyzed. Fluorescent probes were used to assess mitochondrial membrane potential (JC-1), reactive oxygen species (ROS), and lipid peroxidation (LPO). Histological and histomorphometric evaluations were employed to examine tissue structure. SSV better preserved mitochondrial function (JC-1 ratio: 0.64 ± 0.05 vs. 0.43 ± 0.02 in ASF) and reduced ROS (0.56 ± 0.04 vs. 0.96 ± 0.06). CG showed highest LPO (77.15 ± 0.77). ASF had higher scores for spermatogonia/Sertoli distinction (2.26 ± 0.10 vs. 1.30 ± 0.09), nuclear visualization (2.73 ± 0.08 vs. 1.26 ± 0.10), and condensation (2.40 ± 0.09 vs. 1.30 ± 0.09), but smaller tubule diameter (119.58 ± 4.45 vs. 150.82 ± 5.19 μm) and greater basal membrane retraction (48.21 ± 2.36 vs. 32.64 ± 2.16 μm). SSV is superior for canine testicular tissue cryopreservation based on mitochondrial function, oxidative stress, and structural integrity.