Improving porcine SCNT efficiency by selecting donor cells size

Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 μm), group M (medium, 13 μm 18 μm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 μm are more suitable donor cells for SCNT in pigs. <b>Abbreviations</b>: DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.

近年来，猪体细胞核移植（Somatic cell nuclear transfer, SCNT）技术已取得显著进展，但克隆效率仍处于较低水平。供体细胞尺寸可能在体细胞核移植过程中发挥关键作用，但其对猪体细胞核移植的影响尚未明确。本研究旨在通过筛选适宜尺寸的供体细胞，评估猪体细胞核移植的效率。本研究将猪胎儿成纤维细胞（porcine fetal fibroblasts, PFFs）分为三组：S组（小尺寸组，直径d ≤ 13 μm）、M组（中尺寸组，13 μm < d ≤ 18 μm）与L组（大尺寸组，d ≥ 18 μm），并对各组细胞的生物学特性进行分析。随后，采用不同尺寸的PFFs作为供体细胞开展体细胞核移植实验，以评估重构胚胎的发育潜能。研究数据显示，S、M、L三组细胞分别占总细胞数的17.5%、47.7%与34.8%。形态学观察表明，S组细胞的细胞膜与细胞核清晰规整；而M组与L组细胞则出现不同程度的细胞膜隆起、核固缩、自噬及线粒体异常。此外，S组细胞的生长状态与增殖能力显著优于M组与L组；S组与M组的G0/G1期细胞占比显著高于L组；S组细胞的衰老率低于M组与L组；S组细胞的凋亡率显著低于L组，与M组无显著差异。S组的卵裂率同样显著高于M组，与L组无显著差异；S组的囊胚率显著高于M组与L组；S组囊胚的细胞数也显著多于M组与L组。上述研究结果表明，直径小于13 μm的小型猪胎儿成纤维细胞更适合作为猪体细胞核移植的供体细胞。<b>缩略语说明</b>：DMEM：杜尔贝科改良伊格尔培养基（Dulbecco’s modified Eagle’s medium）；FBS：胎牛血清（Fetal bovine serum）；PBS：磷酸盐缓冲液（Phosphate buffer saline）；PFFs：猪胎儿成纤维细胞（Porcine fetal fibroblast cells）；SCNT：体细胞核移植（Somatic cell nuclear transfer）。