Cryopreservation of flushed epididymal spermatozoa outperforms epididymal tail vitrification in Spix's yellow-toothed cavy (Galea spixii)

Galea spixii are important environmental engineers whose reproduction has been affected by habitat fragmentation. The preservation of epididymal spermatozoa emerges as strategy for their conservation. This study aimed to compare in situ vitrification of spermatozoa within the epididymal tail with cryopreservation of flushed epididymal spermatozoa. Epididymal tails from five individuals were allocated into two groups: flushed spermatozoa, which were recovered, cryopreserved in Tris-egg yolk extender with glycerol, stored in liquid nitrogen, thawed and analyzed; and the in situ group, in which epididymal tails were vitrified in Dulbecco's Modified Eagle Medium supplemented with ethylene glycol, stored in liquid nitrogen and rewarmed, followed by cryoprotectant removal and sperm recovery. Samples from fresh and vitrified epididymal tissues were processed for histology and evaluated. Vitrified epididymal tissues presented plasma membrane ruptures, basement membrane retraction, and irregular and non-cohesive luminal content. These injuries significantly impacted post-thawing sperm quality. The flushed spermatozoa group showed higher (P < 0.05) total motility (33.2 ± 4.5%), progressive motility (2.0 ± 0.6%), amplitude of lateral head (7.7 ± 0.8 μm) and rapid sperm population (8.2 ± 2.4%) compared to the in situ group (0.2 ± 0.2%, 0.0 ± 0.0%, 1.9 ± 1.9 μm and 0.0 ± 0.0%, respectively). In conclusion, cryopreservation of flushed epididymal spermatozoa is more effective than epididymal tail vitrification and represents a more suitable strategy for germplasm banking in G. spixii.