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

IF 1.6 3区 农林科学 Q2 AGRICULTURE, DAIRY & ANIMAL SCIENCE
Juliana de Souza Fernandes, Jéssyka Araújo Noronha, Francisco Denilson Rodrigues Gomes, Bruna Farias Brito, Gisele Karla Sena Guimarães, Herlon Victor Rodrigues Silva, Leda Maria Costa Pereira Bersano, Lúcia Daniel Machado da Silva
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引用次数: 0

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.

三种低温保护剂和两种温度组合对玻璃化犬睾丸细胞形态、形态计量学和线粒体活性的影响。
睾丸玻璃化所需的高浓度冷冻保护剂会导致细胞中毒。为了缓解这个问题,一个合适的替代方案是结合冷冻保护剂。加热玻璃化样品的温度对保证细胞活力也很重要。因此,本研究的目的是评估冷冻保护剂(乙二醇- eg、甘油- gly和二甲亚砜- dmso)成对组合并使用两种加热温度(37°C和50°C)对狗睾丸玻璃化后细胞形态、管状形态测定和线粒体活性的影响。将10只成年犬睾丸碎片按冷冻保护剂和温度组合(EG/GLY37、EG/GLY50、DMSO/GLY37、DMSO/GLY50、DMSO/EG37和DMSO/EG50)分为新鲜对照组(CTR)和实验组。将碎片以5.6 mol/L的终浓度(每种冷冻保护剂的2.8 mol/L的2 / 2组合)玻璃化,然后加热到37°C/30 s或50°C/5 s。随后,用罗丹明123对它们进行组织形态学、形态计量学和线粒体活性评估。在形态学评估中,所有玻璃化组均显示管状直径显著减小(p < 0.05)。关于精原细胞和支持细胞的区别,除DMSO/GLY37外,加热至50°C时,没有组与对照组不同。对于核可视化,除DMSO/GLY37 (p 0.05)外,玻璃化组与CTR均无差异(p 0.05)。在所有的玻璃化样品中,无论冷冻保护剂和加热温度的组合如何,线粒体活性都降低(p > 0.05)。由此可见,DMSO/GLY50的结合在形态学方面具有较好的保存效果。
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来源期刊
Reproduction in Domestic Animals
Reproduction in Domestic Animals 农林科学-奶制品与动物科学
CiteScore
3.00
自引率
5.90%
发文量
238
审稿时长
4-8 weeks
期刊介绍: The journal offers comprehensive information concerning physiology, pathology, and biotechnology of reproduction. Topical results are currently published in original papers, reviews, and short communications with particular attention to investigations on practicable techniques. Carefully selected reports, e. g. on embryo transfer and associated biotechnologies, gene transfer, and spermatology provide a link between basic research and clinical application. The journal applies to breeders, veterinarians, and biologists, and is also of interest in human medicine. Interdisciplinary cooperation is documented in the proceedings of the joint annual meetings. Fields of interest: Animal reproduction and biotechnology with special regard to investigations on applied and clinical research.
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