Lima David Baruc Cruvinel, Silva Ticiana Franco Pereira da, Aquino-Cortez Annice, Leiva-Revilla Johanna, Silva Lúcia Daniel Machado da
Laboratory of Carnivore Reproduction, School of Veterinary Medicine, State University of Ceará (Universidade Estadual do Ceará, UECE) - 1700, Doutor Silas Munguba Avenue, CEP 60714-903, Fortaleza, CE, Brazil.
Laboratory of Carnivore Reproduction, School of Veterinary Medicine, State University of Ceará (Universidade Estadual do Ceará, UECE) - 1700, Doutor Silas Munguba Avenue, CEP 60714-903, Fortaleza, CE, Brazil.
Theriogenology. 2018 Apr 1;110:110-115. doi: 10.1016/j.theriogenology.2017.12.037. Epub 2018 Jan 5.
Protocols for the cryopreservation of testicular tissue are not yet established. In cats, few studies have been conducted on testicular vitrification using different cryoprotectant associations (CPAs). Thus, the objective of this study was to compare the effect of different CPAs on the vitrification of testicular tissue from prepubertal cats in cryotubes. We used 10 pairs of testicles, with each pair divided into 8 fragments that were distributed into different experimental groups. Two of these fragments were allocated into the control group (CG) and the other six were distributed according to the CPAs to be tested (dimethyl sulfoxide (DMSO)/glycerol (GLY), ethylene glycol (EG)/GLY, or DMSO/EG). The cryoprotectants were used at a final concentration of 5.6 M. The fragments were subjected to vitrification in cryotubes and after 1 week, they were warmed and processed for histomorphologic assessment, quantification of nucleolar organizer regions (NORs), and determination of cell viability. The DMSO/EG and EG/GLY groups presented the greatest cell separation from the cell basement membrane and the highest degrees of retraction of the basal membrane. In these aspects, DMSO/GLY did not differ from the CG and both were significantly superior to the other groups. In terms of cell distinction, visibility of the nucleus, and nuclear condensation, all the vitrified groups had significantly lower values than the CG, while the DMSO/GLY and EG/GLY groups did not differ between themselves. Through the quantification of NORs, the potential for cell proliferation of the CG was found to have a mean of 3.80, while DMSO/GLY presented a mean of 3.60, and thus there was no significant difference between these two groups. The proliferation potentials of both groups were significantly superior to that of the DMSO/EG (mean: 2.07) and EG/GLY (mean: 1.98) groups. In the CG and DMSO/GLY group, 91.8% and 64.2% of cells, respectively, were found to be viable. The cell viabilities of both groups were significantly superior to those of DMSO/EG (52.5%) and EG/GLY (57.10%). Vitrification in cryotubes combined with the use of the DMSO/GLY association was effective in maintaining the histomorphology, cell proliferation potential, and cell viability of testicular tissue from prepubertal cats after cryopreservation.
睾丸组织冷冻保存的方案尚未确立。在猫身上，关于使用不同冷冻保护剂组合（CPA）进行睾丸玻璃化的研究较少。因此，本研究的目的是比较不同CPA对青春期前猫睾丸组织在冷冻管中玻璃化的影响。我们使用了10对睾丸，每对分成8个片段，分别分配到不同的实验组。其中2个片段被分配到对照组（CG），另外6个根据要测试的CPA进行分配（二甲亚砜（DMSO）/甘油（GLY）、乙二醇（EG）/GLY或DMSO/EG）。冷冻保护剂的终浓度为5.6 M。将片段在冷冻管中进行玻璃化处理，1周后，进行复温并进行组织形态学评估、核仁组织区（NOR）定量以及细胞活力测定。DMSO/EG组和EG/GLY组出现了最大程度的细胞与细胞基底膜分离以及基底膜最高程度的回缩。在这些方面，DMSO/GLY组与CG组无差异，且两者均显著优于其他组。在细胞区分、细胞核可见性和核浓缩方面，所有玻璃化组的值均显著低于CG组，而DMSO/GLY组和EG/GLY组之间无差异。通过NOR定量发现，CG组的细胞增殖潜力平均值为3.80，而DMSO/GLY组平均值为3.60，因此这两组之间无显著差异。这两组的增殖潜力均显著优于DMSO/EG组（平均值：2.07）和EG/GLY组（平均值：1.98）。在CG组和DMSO/GLY组中，分别发现91.8%和64.2%的细胞具有活力。这两组的细胞活力均显著优于DMSO/EG组（52.5%）和EG/GLY组（57.10%）。冷冻管中的玻璃化结合DMSO/GLY组合使用，在冷冻保存后能有效维持青春期前猫睾丸组织的组织形态学、细胞增殖潜力和细胞活力。
