Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.

中文翻译：

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来自猫科动物的分离睾丸细胞冷冻保存后的细胞存活率

睾丸细胞悬液 (TCS) 可以冷冻保存，用于男性生殖系保存和生育力恢复。我们旨在验证用于濒危猫科动物的家猫 TCS 冷冻保存方案。来自成年家猫的睾丸组织被酶解并富集了生精细胞。将所得 TCS 稀释在 7.5% 或 15% Me2SO 基培养基中。测试了慢速和快速冷冻方法。我们使用两种荧光染料组合检查了冷冻方法的效果：钙黄绿素-AM 与碘化丙啶 (C/PI) 和 SYBR14 与碘化丙啶 (S/PI)。家猫新鲜TCS的倍性分析表明，大多数睾丸细胞是单倍体细胞。基于显微观察，两个大小种群（12.3 ± 2.3 μm 和 20. 直径为 5 ± 4 μm）被鉴定并推测分别主要是精子细胞和精母细胞。两种评估方法都证明，与单细胞相比，冷冻保存前后聚集细胞的存活率更高，并且低浓度 Me2SO (7.5%) 与缓慢冷冻相结合以保持睾丸细胞的存活率具有优势。然而，与 C/PI 方法相比，S/PI 导致更精确的评估。7.5% 基于 Me2SO 的培养基与缓慢冷冻的组合产生了 S/PI 标记的聚合 (49.8 ± 20%) 和单细胞 (31.5 ± 8.1%) 的解冻后活力。使用猎豹和亚洲金猫的睾丸获得了类似的结果。总之，来自家猫的 TCS 可以成功冷冻保存，并有可能支持濒危猫科动物物种的生育能力恢复。