Moro L N, Sestelo A J, Salamone D F
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.
Jardín Zoológico de la Ciudad de Buenos Aires, República de la India 3000, Buenos Aires, Argentina.
Reprod Domest Anim. 2014 Aug;49(4):693-700. doi: 10.1111/rda.12355. Epub 2014 Jun 26.
The ICSI procedure is potentially of great value for felids, and it has not been extensively studied in these species. The objectives of this work were to determine the best conditions for ICSI in the domestic cat (DC) to generate interspecific embryos by injecting cheetah (Ch) and leopard (Leo) spermatozoa. Firstly, DC oocytes were matured with insulin-transferrin-selenium (ITS) or without it (MM) and cultured using atmospheric (21%) or low (5%) oxygen tension after ICSI. The group ITS-5%O2 showed the highest blastocyst rate (p < 0.05), 20.9% vs 8.7%, 7% and 6.5%, for MM-21%O2 , MM-5%O2 and ITS-21%O2 , respectively. The best conditions were used to generate the interspecific embryos, together with ionomycin activation (Io) after ICSI. Interspecific embryos resulted in high rates of blastocysts that were not positively affected by Io activation: 32.6% vs 21% for Ch and Ch-Io, 9.8% vs 21% for Leo and Leo-Io, and 20% vs 17.4% for DC and DC-Io. We also evaluated DNA-fragmented nuclei of experiment 1 and 2 blastocysts, using TUNEL assay. The fragmented nucleus proportion was higher in the ITS-5%O2 group, 67.6%. Surprisingly, interspecific blastocysts showed the lowest fragmented nucleus proportion: 27% and 29.9% for Ch and Leo, respectively. We concluded that ITS and 5%O2 improve blastocyst formation in DC, although with a concomitant increase in DNA fragmentation. Most importantly, cheetah and leopard spermatozoa were able to generate blastocysts without artificial activation, which suggests that developmental capacity of wild felid spermatozoa can be evaluated by interspecific ICSI. This technique should be used to assist wild felid reproduction.
卵胞浆内单精子注射（ICSI）技术对猫科动物可能具有巨大价值，但尚未在这些物种中得到广泛研究。本研究的目的是确定家猫（DC）进行ICSI的最佳条件，通过注射猎豹（Ch）和豹（Leo）的精子来产生种间胚胎。首先，DC卵母细胞在有胰岛素 - 转铁蛋白 - 硒（ITS）或无ITS（MM）的条件下成熟，并在ICSI后分别使用大气氧浓度（21%）或低氧浓度（5%）进行培养。ITS - 5%O₂组的囊胚率最高（p < 0.05），分别为20.9%，而MM - 21%O₂、MM - 5%O₂和ITS - 21%O₂组的囊胚率分别为8.7%、7%和6.5%。将最佳条件用于产生种间胚胎，并在ICSI后联合使用离子霉素激活（Io）。种间胚胎产生了较高比例的囊胚，且Io激活对其没有积极影响：Ch和Ch - Io组分别为32.6%和21%，Leo和Leo - Io组分别为9.8%和21%，DC和DC - Io组分别为20%和17.4%。我们还使用末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）评估了实验1和2中囊胚的DNA片段化细胞核。ITS - 5%O₂组的细胞核片段化比例较高，为67.6%。令人惊讶的是，种间囊胚的细胞核片段化比例最低：Ch和Leo分别为27%和29.9%。我们得出结论，ITS和5%O₂可提高DC的囊胚形成率，尽管同时会增加DNA片段化。最重要的是，猎豹和豹的精子能够在无需人工激活的情况下产生囊胚，这表明野生猫科动物精子的发育能力可通过种间ICSI进行评估。该技术应用于辅助野生猫科动物繁殖。
