While intracytoplasmic sperm injection (ICSI) is an asset in human Assisted Reproduction Technologies (ART), its outcomes, in terms of blastocyst, is still unacceptably low in ruminants. The picture typically found in ICSI derived bovine and ovine embryos is an asymmetry between a high activation rate, marked by a pronuclear development, and a low first cleavage rate. Abnormal centriole function has been indicated as a possible factor which undermines embryonic development following ICSI, especially when Freeze Dried spermatozoa (FD) are used. In order to verify the hypothesis that centriole dysfunction might be responsible for low ICSI outcomes in sheep, we have investigated micro-tubular dynamics, markedly aster nucleation, in fertilized sheep zygotes by ICSI with frozen/thawed (FT) and FD spermatozoa; In Vitro Fertilized (IVF) sheep oocytes were used as control. The spermatozoa aster nucleation was assessed at different time points following ICSI and IVF by immune-detection of α-tubulin. Pronuclear stage, syngamy and embryo development were assessed. No difference was noticed in the timing of aster nucleation and microtubule elongation in ICSI-FT derived embryos with control IVF ones, while a delay was recorded in ICSI-FD ones. The proportion of 2-pronuclear stage zygotes was similar in ICSI-FT and ICSI-FD (47% and 53%, respectively), both much lower comparing the IVF ones (73%). Likewise, syngamy was observed in a minority of both ICSI groups (28.5% vs 12.5% in ICSI-FT/FD respectively) comparing to IVF controls (50%), with a high number of zygotes blocked at the 2-pronuclear stage (71.5% vs 87.5% respectively). While no significant differences were noticed in the cleavage rate between ICSI-FD, ICSI-FT and IVF groups (31%, 34% and 44%) respectively, development to blastocyst stage was markedly compromised in both ICSI groups, especially with FD spermatozoa (10% in ICIS-FD and 19% in ICSI-FT vs 33% in IVF (P < 0.005, ICSI-FD vs IVF and P < 0.05, IVF vs ICSI-FT, respectively). Hence, here we have demonstrated that the reduced cleavage, and the ensuing impaired development to blastocysts stage of ICSI derived sheep embryos is not related to centriole dysfunction, as suggested by other authors. The major recorded problem is the lack of syngamy in ICSI derived zygotes, an issue that should be addressed in further studies to improve ICSI procedure in sheep embryos.

中文翻译：

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绵羊ICSI衍生胚胎发育受损与中心粒功能障碍无关

虽然卵胞浆内单精子注射 (ICSI) 是人类辅助生殖技术 (ART) 的一项资产，但就囊胚而言，其结果在反刍动物中仍然低得令人无法接受。通常在 ICSI 衍生的牛和绵羊胚胎中发现的图片是高激活率（以原核发育为标志）和低第一次卵裂率之间的不对称。异常的中心粒功能已被认为是破坏 ICSI 后胚胎发育的一个可能因素，尤其是当使用冻干精子 (FD) 时。为了验证中心粒功能障碍可能导致绵羊 ICSI 结果低的假设，我们研究了具有冷冻/解冻 (FT) 和 FD 精子的 ICSI 受精绵羊受精卵的微管动力学，尤其是紫菀成核；体外受精 (IVF) 绵羊卵母细胞用作对照。在 ICSI 和 IVF 后的不同时间点通过 α-微管蛋白的免疫检测评估精子星形成核。评估原核阶段、配偶和胚胎发育。在 ICSI-FT 衍生胚胎与对照 IVF 胚胎中，紫菀成核和微管伸长的时间没有差异，而在 ICSI-FD 胚胎中记录到延迟。ICSI-FT 和 ICSI-FD 中 2 原核阶段受精卵的比例相似（分别为 47% 和 53%），与 IVF 的比例（73%）相比都低得多。同样，与 IVF 对照 (50%) 相比，在两个 ICSI 组中的少数 (ICSI-FT/FD 中分别为 28.5% 和 12.5%) 中观察到同配，大量受精卵在 2-原核阶段被阻断 (71.5 % vs 87.5%）。ICSI-FD 与 IVF 和 P < 0.05，IVF 与 ICSI-FT，分别）。因此，在这里我们已经证明，正如其他作者所建议的那样，ICSI 衍生的绵羊胚胎的卵裂减少以及随之而来的胚泡阶段发育受损与中心粒功能障碍无关。记录的主要问题是 ICSI 衍生的受精卵缺乏同配，这个问题应该在进一步研究中解决，以改进绵羊胚胎的 ICSI 程序。ICSI-FD 与 IVF 和 P < 0.05，IVF 与 ICSI-FT，分别）。因此，在这里我们已经证明，正如其他作者所建议的那样，ICSI 衍生的绵羊胚胎的卵裂减少以及随之而来的胚泡阶段发育受损与中心粒功能障碍无关。记录的主要问题是 ICSI 衍生的受精卵缺乏同配，这个问题应该在进一步研究中解决，以改进绵羊胚胎的 ICSI 程序。