Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, D-10315, Berlin, Germany.
Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, D-10315, Berlin, Germany.
Cryobiology. 2020 Dec;97:191-197. doi: 10.1016/j.cryobiol.2020.03.001. Epub 2020 Mar 16.
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.
睾丸细胞悬液 (TCS) 可冷冻保存用于雄性生殖系保存和生育力恢复。我们旨在验证一种用于冷冻保存家猫 TCS 的方案，以便应用于濒危猫科动物物种。从成年家猫的睾丸组织中酶解分离生精细胞，然后进行富集。所得 TCS 在 7.5%或 15% Me2SO 基础培养基中稀释。测试了慢速冻和快速冻方法。我们使用两种荧光染料组合（Calcein-AM 与碘化丙啶（C/PI）和 SYBR14 与碘化丙啶（S/PI））来检查冷冻方法的效果。对家猫新鲜 TCS 的倍性分析表明，大多数睾丸细胞是单倍体细胞。根据显微镜观察，确定了两个大小群体（直径分别为 12.3±2.3μm 和 20.5±4μm），并分别推测主要是精母细胞和精细胞。两种评估方法均证明，与单细胞相比，冷冻前和冷冻后聚集细胞的活力更高，并且在慢速冻过程中使用低浓度 Me2SO（7.5%）比保存睾丸细胞活力更具优势。然而，与 C/PI 方法相比，S/PI 方法的评估结果更准确。7.5% Me2SO 基础培养基与慢速冻相结合，可使 S/PI 标记的聚集（49.8±20%）和单个细胞（31.5±8.1%）在解冻后具有活力。使用猎豹和亚洲金猫的睾丸也得到了类似的结果。总之，家猫的 TCS 可以成功冷冻保存，并有潜力支持濒危猫科动物物种的生育力恢复。
