Noronha Jéssyka Araújo, de Souza Fernandes Juliana, Gomes Francisco Denilson Rodrigues, Colares Julia Carrah, Guimarães Gisele Karla Sena, Silva Herlon Victor Rodrigues, da Silva Lúcia Daniel Machado
Laboratório de Reprodução de Carnívoros (LRC), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.
Laboratório de Manipulação de Oócitos e Folículos Ovarianos Pré-Antrais (LAMOFOPA), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.
Reprod Domest Anim. 2025 Jun;60(6):e70081. doi: 10.1111/rda.70081.
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.
睾丸玻璃化冷冻需要使用高浓度的冷冻保护剂，由于其毒性，这些冷冻保护剂会对样本造成损害。这些物质的组合被提出作为减轻这一问题的一种方法。因此，本研究的目的是评估三种冷冻保护剂组合在犬睾丸玻璃化冷冻中的效果。使用了来自成年犬的10对睾丸，从每对睾丸中获取12个片段，根据所测试的冷冻保护剂组合分配到新鲜对照组（CTR）和实验组：二甲亚砜（DMSO）/乙二醇（EG）、DMSO/甘油（GLY）和EG/GLY。采用固体表面玻璃化法（SSV）对片段进行玻璃化冷冻，冷冻保护剂组合的最终浓度为5.6 mol/L。随后，将它们复温并进行组织形态计量学评估，并用罗丹明123测定线粒体活性。考虑到形态学评估，DMSO/EG组的结果与CTR组相似，生精小管中的核完整性和细胞组织评分良好（p > 0.05）。相比之下，EG/GLY组出现了更大程度的核浓缩。很难观察到精原细胞和支持细胞并将它们区分开来（p < 0.05）。DMSO/GLY组在精原细胞和支持细胞之间也表现出明显差异，以及核浓缩，这在统计学上与CTR组不同（p < 0.05）。此外，在EG/GLY组和DMSO/GLY组的生精小管中观察到剩余细胞的随机分布。三个测试组均出现基底膜回缩，生精小管平均直径减少约11.6%（p < 0.05）。无论使用何种冷冻保护剂组合，玻璃化冷冻均未影响样本的线粒体活性（p > 0.05）。得出的结论是，DMSO/EG组合最有助于维持犬睾丸玻璃化冷冻后的组织形态结构。
