Evaluation of Different Cryoprotectant Combinations in Testicular Vitrification in Dogs

Abstract

Testicular vitrification requires the use of high concentrations of cryoprotectants, which can cause damage to samples due to their toxicity. The combination of these substances comes up as a way to mitigate this problem. Thus, the aim of this study was to evaluate three cryoprotectant combinations in the testicular vitrification of dogs. Ten testicular pairs from adult dogs were used, from which 12 fragments of each pair were obtained, distributed among the fresh control group (CTR) and the experimental groups according to the cryoprotectant combinations tested: dimethyl sulfoxide (DMSO)/ethylene glycol (EG), DMSO/glycerol (GLY), and EG/GLY. The fragments were vitrified using the solid surface vitrification method (SSV), at a final concentration of 5.6 mol/L of the combined cryoprotectants. Subsequently, they were warmed up and processed for histomorphological morphometric evaluations and determination of mitochondrial activity with Rhodamine 123. Considering the morphological evaluation, the DMSO/EG group showed results similar to CTR, with good scores for nuclear integrity and cell organisation in the seminiferous tubules (p > 0.05). In contrast, the EG/GLY group presented greater nuclear condensation. It was difficult to visualise and distinguish between spermatogonia and Sertoli cells (p < 0.05). The DMSO/GLY group also showed distinct levels between spermatogonia and Sertoli cells, as well as nuclear condensation, which statistically differed from CTR (p < 0.05). Also, it was observed a random distribution of the remaining cells in the seminiferous tubules of the EG/GLY and DMSO/GLY groups. The three tested groups showed basement membrane retraction and a reduction of approximately 11.6% in the average diameter of the seminiferous tubules (p < 0.05). Vitrification did not influence the mitochondrial activity of the samples, regardless of the combination of cryoprotectants used (p > 0.05). It was concluded that the DMSO/EG combination best contributed to the maintenance of the testicular histomorphological structure of dogs after vitrification.

Keywords: cryopreservation; dimethyl sulfoxide; ethylene glycol; glycerol; testicles.

FIGURE 1

Experimental design. Distribution of experimental groups according to cryoprotectant combinations and evaluation techniques.

FIGURE 2

Histomorphological evaluation of canine testicular fragments. (A) Fresh control group (CTR); (B) DMSO/EG group showing good integrity; (C) DMSO/GLY group with cellular loss and basement membrane separation (asterisks); (D) EG/GLY group showing disorganised tubular structure and difficult distinction between spermatogonia (arrowheads) and Sertoli cells (arrows). (HE, 400x, scale: 10 μm).

FIGURE 3

Photomicrograph illustrating the reduction in tubular diameter after vitrification. (A) Fresh control group; (B) DMSO/EG group. (HE, 200x, scale: 50 μm).

FIGURE 4

Mean ± SD of the diameter of canine seminiferous tubules. Fresh control group (CTR) and vitrified groups with different cryoprotectant combinations: DMSO/EG, DMSO/GLY, and EG/GLY, respectively. Lowercase letters superscript differently on the same line indicate statistical differences among groups (p < 0.05).

FIGURE 5

Photomicrographs of testicular fragments demonstrating mitochondrial activity under fluorescent microscopic analysis with Rhodamine 123. (A) Fresh control group (CTR); (B) DMSO/EG; (C) DMSO/GLY; (D) EG/GLY. (40x; scale: 100 μm).

FIGURE 6

Evaluation of the viability of canine testicular fragments, fresh control group (CTR) and vitrified groups with different cryoprotectant combinations: DMSO/EG, DMSO/GLY, and EG/GLY. Lowercase letters superscript differently on the same line indicate statistical differences between groups (p < 0.05).