# Influence of Three Combinations of Cryoprotectants and Two Warming Temperatures on Cellular Morphology, Morphometry and Mitochondrial Activity of Vitrified Canine Testicles

## TL;DR

This study examines how different cryoprotectant combinations and warming temperatures affect the structure and mitochondrial activity of vitrified canine testicles.

## Contribution

The study introduces a novel evaluation of cryoprotectant combinations and warming temperatures for preserving canine testicular tissue morphology and mitochondrial activity.

## Key findings

- Combining DMSO and glycerol with a 50°C warming temperature preserved morphological aspects better than other combinations.
- All vitrified groups showed reduced mitochondrial activity compared to the control.
- DMSO/GLY50 showed no significant differences in nuclear visualization compared to the control.

## Abstract

High concentrations of cryoprotectants required for testicular vitrification result in a toxic environment for cells. To mitigate this issue, a suitable alternative is to combine cryoprotectants. The temperature for warming a vitrified sample is also important to assure cell viability. Thus, the aim of this work was to evaluate how combining cryoprotectants (ethylene glycol‐EG, glycerol‐GLY, and dimethyl sulfoxide‐DMSO) in pairs and using two warming temperatures (37°C and 50°C) influence cellular morphology, tubular morphometry, and mitochondrial activity after testicular vitrification of dogs. Testicular fragments from ten adult dogs were distributed among the fresh control group (CTR) and the experimental groups according to the combination of cryoprotectants and temperatures (EG/GLY37, EG/GLY50, DMSO/GLY37, DMSO/GLY50, DMSO/EG37 and DMSO/EG50). The fragments were vitrified in a final concentration of 5.6 mol/L (2.8 mol/L of each of the cryoprotectants combined two by two) and subsequently warmed up to 37°C/30 s or 50°C/5 s. Following this, they were processed for histomorphological, morphometric, and mitochondrial activity evaluations with Rhodamine 123. In the morphometric evaluation, all vitrified groups showed a significant reduction in tubular diameter (p < 0.05). All experimental groups showed greater basement membrane separation when related to the CTR (p < 0.05). DMSO/EG37 showed the greatest basement membrane separation when compared to all other groups (p < 0.05). Regarding membrane retraction, all vitrified groups, regardless of the warming temperature, had greater retraction when related to CTR (p < 0.05), except DMSO/GLY50, which did not differ from any group (p > 0.05). Regarding the distinction between spermatogonia and Sertoli cells, no groups warmed up to 50°C differed from the control, except DMSO/GLY37. For nuclear visualisation, none of the vitrified groups differed from the CTR (p > 0.05), except DMSO/GLY37 (p < 0.05), which showed better nuclear visualisation. For the nuclear condensation parameter, there were no significant differences among the groups (p > 0.05). Mitochondrial activity was reduced in all vitrified samples, regardless of the combination of cryoprotectants and warming temperature (p > 0.05). It was concluded that the association of DMSO/GLY50 presented better preservation of morphological aspects.