Cryopreservation of Testicular Tissue from Adult Red-Rumped Agoutis (Dasyprocta leporina Linnaeus, 1758)

Abstract

This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm³) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface vitrification (SSV), and conventional vitrification (CV). Each method used dimethyl sulfoxide (DMSO), ethylene glycol (EG), or a DMSO + EG combination. Morphology, viability, mitochondrial activity, and proliferative potential were assessed in fresh and frozen tissue samples. Testicular morphology was better using SSV with a combination of DMSO and EG. Across the different cryopreservation approaches, as well as cryoprotectant combinations, cell viability was comparable. Regarding mitochondrial activity, DMSO + EG/SSV or CV, and DMSO + EG/CV were similar to the EG/SF group, which was the best group that provided values similar to fresh control groups. Adequate preservation of the proliferative potential of spermatogonia, Leydig cells, and Sertoli cells was obtained using SSV with DMSO + EG. Overall, the use of SSV with DMSO + EG was the best protocol for the preservation of testicular tissues from adult red-rumped agoutis.

Keywords: biobanking; permeating cryoprotectants; testicular tissue; vitrification; wild rodents.

Figure 1

Representative micrographs used for histomorphological evaluations of testicular tissue sections from adult red-rumped agoutis (n = 5 males) after different cryopreservation treatments. (A) Non-cryopreserved control group, and cryopreserved groups using (B) slow freezing [SF] with dimethyl sulfoxide [DMSO], (C) SF with ethylene glycol [EG], (D) SF with DMSO + EG, (E) solid-surface vitrification [SSV] with DMSO, (F) SSV with EG, (G) SSV with EG + DMSO, (H) conventional vitrification [CV] with DMSO, (I) CV with EG, (J) CV with DMSO + EG. Black arrows indicate rupture from the basal membrane. White arrow shows shrinkage from the basal membrane. Asterisks shows tubular cell loss. White arrowheads show cell swelling. Scale bar: 20 µm.

Figure 2

Evaluations of cell viability in testicular tissues from adult red-rumped agoutis (n = 5 males) after different cryopreservation treatments. (A) Representative picture of testicular cell viability evaluated by fluorescent probes. Blue arrow indicates cells in suspension stained with Hoechst 33342; red arrow indicates non-viable cells in the same suspension stained with propidium iodide. (B) Average proportions (mean ± SEM) of viable cells in testicular tissues exposed to different treatments (slow freezing, SF; conventional vitrification, CV; and solid-surface vitrification, SSV) and different cryoprotectants: dimethyl sulfoxide (DMSO), ethylene glycol (EG), and DMSO + EG. Scale bar: 20 µm. ^a,b Different lowercase letters above bars indicate differences among treatments (p < 0.05).

Figure 3

Evaluations of mitochondrial activity (MitoTracher Red staining) in testicular tissues from adult red-rumped agoutis (n = 5) after different cryopreservation treatments. (A) Representative images for: (a) Hoechst 33342 staining, (b) MitoTracher Red staining, (c) merged images; (B) Average values (mean ± SEM) of arbitrary unities used for the evaluation of mitochondrial activity in testicular tissues exposed to different treatments (slow freezing, SF; conventional vitrification, CV; and solid-surface vitrification, SSV) and different cryoprotectants: dimethyl sulfoxide (DMSO), ethylene glycol (EG), and DMSO + EG. Scale bar: 20 µm. ^a–e Different lowercase letters above bars indicate differences among treatments (p < 0.05).

Figure 4

(A) Representative picture of proliferative potential evaluated by the quantification of nucleolar organizer regions (NORs) in testicular cells from adult red-rumped agoutis (n = 5 males): (a) Seminiferous tubule, scale bar: 20 µm; (b) enlarged seminiferous tubule area showing spermatogonia (white arrow), Leydig cell (black arrowhead), and Sertoli cell (black arrow), scale bar: 10 µm. (B–D) evaluations of proliferative capacity as the average number NORs (mean ± SEM) in spermatogonia (B), Leydig cells (C), and Sertoli cells (D) exposed to different treatments (slow freezing, SF; solid-surface vitrification, SSV; and conventional vitrification, CV) with different cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG; DMSO + EG combination). ^a–d Different lowercase letters above bars indicate differences among treatments (p < 0.05).