未经授权:睾丸组织的冷冻保存为保护濒危物种提供了新的可能性,遗传上有价值的个体,甚至是意外死亡的青春期前个体的生育潜力。然而,这项技术的使用仍然是一个挑战。在这项研究中,研究了睾丸组织的缓慢冷冻和玻璃化保存方法,以找出哪种冷冻保存方法可以更好地保存多种野生物种的睾丸生殖细胞的活力和DNA完整性。
UNASSIGNED:睾丸是从18只偶蹄动物(野猪,鹿,矮人山羊,麦尔·瞪羚,欧洲Mouflon,非洲森林水牛,马来亚塔米尔,DorcasGazelle,伊比利亚ibex,gnu,红河猪),5只灵长类动物(疣猴,卷尾猴,mandrill),8个食肉动物(灰狼,波斯豹,宾图龙,欧洲水貂,美国黑熊,suricata),和2只啮齿动物(巴塔哥尼亚马拉)。睾丸属于成年人,被切成小块,并通过针头浸入式玻璃化或使用被动冷却装置进行不受控制的缓慢冷冻进行冷冻保存。升温或解冻后,睾丸组织被酶消化,两种生殖细胞类型根据其形态进行分化:圆形细胞(精原细胞,精母细胞,和早期精子)和细长细胞(细长精子和精子)。通过SYBR-14/碘化丙啶评估细胞活力,同时通过荧光显微镜的TUNEL测定进行DNA片段化。
UNASSIGNED:我们的初步结果表明,与玻璃化相比,我们不受控制的缓慢冷冻方法更好地保留了细长细胞的活力和DNA完整性。在所有物种中都观察到这种趋势,在偶蹄动物中很重要,食肉动物,和灵长类动物。同样,通过不受控制的缓慢冷冻,灵长类动物中圆形细胞的活力和DNA完整性也得到了更好的维持,在食肉动物中,通过针浸玻璃化在这种类型的细胞中显示出更好的结果。在偶蹄动物和啮齿动物中,两种技术都以类似的方式保留了圆形细胞的活力,尽管在偶蹄动物中进行针浸玻璃化后,这些细胞的DNA完整性更高。
未经批准:总而言之,每种冷冻保存方法的有效性受到物种和细胞类型之间的系统发育多样性的影响。
UNASSIGNED: Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species.
UNASSIGNED: Testes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope.
UNASSIGNED: Our preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls.
UNASSIGNED: In conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.