Valdivia Martha, Bravo Zezé, Reyes Jhakelin, Gonzales Gustavo F
Laboratory of Reproductive Physiology, Research Institute "Antonio Raimondi," Zoology Department, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, Lima, Peru.
Endocrine and Reproductive Laboratory, Department of Biological and Physiological Science, and Laboratory of Investigation and Development (LID), Faculty of Sciences and Philosophy, Universidad Peruana Cayetano Heredia, Lima, Peru.
Front Vet Sci. 2021 Mar 17;8:597964. doi: 10.3389/fvets.2021.597964. eCollection 2021.
This is the first time that testicular tissue ( = 44) and isolated testicular cells ( = 51) were cryopreserved from alpaca testes 24 h postmortem. For this purpose, internally designed freezing media and cryopreservation protocols were used. Testicular tissue fragments (25 mg) and isolated testicular cells were frozen in MTDB (trehalose and black maca), MTD (trehalose), MSDB (sucrose and black maca), and MSD (sucrose) media. Isolated spermatogonial cells were cryopreserved in two ways, before and after proliferation . After cryopreservation, the percentage of cell viability in Group 1 (>50% of cell viability) by trypan blue did not show differences within each group ( > 0.05) but showed significant differences when comparing fragments with isolated cells ( < 0.05). Spermatogonial stem cells (SSC) were identified by flow cytometry as strong agglutinin (sDBA) and mitochondrial activity of SSC as strongly positive for MitoSense (sMitoSense+) in intact mitochondria cells, weakly positive for MitoSense (wMitoSense+) in early apoptosis, and necrosis with 7-Aminoactinomycin-D positive (7-AAD). After freezing, in Group 1M (≥30% sMitoSense+), the fragments did not show differences between the media ( > 0.05), but in the isolated cells frozen in MSDB medium, 63.68 ± 8.90% ( < 0.05). In Group 2M (<30% sMitoSense+), necrosis (7AAD+) in MSDB medium was 27.03 ± 5.80%, and necrosis in isolated cells was 14.05 ± 9.3% with significant differences between these groups ( < 0.05); in sMitoSense+, the isolated cells (34.40 ± 23%) had a higher percentage than the fragments (12.4 ± 5.2) ( < 0.05). On the other hand, MSDB and MSD media were significantly higher for isolated cells than for fragments in sDBA+ ( < 0.05). On the other hand, the SSC (sDBA+) had significant differences ( < 0.05) between fresh cells 7.43 ± 1.3% (sDBA+) compared with those cryopreserved in MSDB medium 1.46 ± 0.34% (sDBA+). Additionally, the proliferated and cryopreserved SSC 6.29 ± 1.17% (sDBA+) did not show significant differences concerning the fresh cells ( > 0.05). In conclusion, the black maca showed antioxidant properties when it was included in the freezing medium and, therefore, improved the SSC's conservation of the alpaca. Furthermore, the proliferation of isolated cells produces a higher amount of SSC after thawing them for further preclinical or clinical work.
这是首次在羊驼死后24小时从其睾丸中冷冻保存睾丸组织（n = 44）和分离的睾丸细胞（n = 51）。为此，使用了内部设计的冷冻培养基和冷冻保存方案。将睾丸组织碎片（25毫克）和分离的睾丸细胞分别在MTDB（海藻糖和黑玛卡）、MTD（海藻糖）、MSDB（蔗糖和黑玛卡）和MSD（蔗糖）培养基中冷冻。分离的精原细胞通过两种方式进行冷冻保存，即增殖前后。冷冻保存后，通过台盼蓝检测，第1组（细胞活力>50%）的细胞活力百分比在每组内无差异（P>0.05），但与分离细胞相比，组织碎片显示出显著差异（P<0.05）。通过流式细胞术鉴定精原干细胞（SSC）为强凝集素（sDBA）阳性，完整线粒体细胞中SSC的线粒体活性为MitoSense强阳性（sMitoSense+），早期凋亡时为MitoSense弱阳性（wMitoSense+），坏死时为7-氨基放线菌素-D阳性（7-AAD）。冷冻后，在第1M组（≥30% sMitoSense+）中，组织碎片在不同培养基之间无差异（P>0.05），但在MSDB培养基中冷冻的分离细胞中，该比例为63.68±8.90%（P<0.05）。在第2M组（<30% sMitoSense+）中，MSDB培养基中的坏死率（7AAD+）为27.03±5.80%，分离细胞中的坏死率为14.05±9.3%，两组间差异显著（P<0.05）；在sMitoSense+方面，分离细胞（34.40±23%）的比例高于组织碎片（12.4±5.2）（P<0.05）。另一方面，在sDBA+方面，MSDB和MSD培养基中分离细胞的比例显著高于组织碎片（P<0.05）。另一方面，新鲜细胞中SSC（sDBA+）的比例为7.43±1.3%，与MSDB培养基中冷冻保存的细胞（1.46±0.34%，sDBA+）相比有显著差异（P<0.05）。此外，增殖并冷冻保存的SSC（6.29±1.17%，sDBA+）与新鲜细胞相比无显著差异（P>0.05）。总之，当黑玛卡包含在冷冻培养基中时，它表现出抗氧化特性，因此改善了羊驼精原干细胞的保存。此外，分离细胞的增殖在解冻后产生了更多的精原干细胞，可用于进一步的临床前或临床工作。
