Key Lab of Animal Cell Technology, National Institute of Animal Science, Hanoi, Viet Nam.
Key Lab of Animal Cell Technology, National Institute of Animal Science, Hanoi, Viet Nam.
Theriogenology. 2024 Dec;230:115-120. doi: 10.1016/j.theriogenology.2024.09.013. Epub 2024 Sep 14.
We aimed to establish efficient donor cells to produce piglets by somatic cell nuclear transfer (SCNT) of the endangered Vietnamese Ỉ pig. In Experiment 1, we assessed the effects of cell passages on the in vitro development of SCNT embryos. Cells with five and six passages showed significantly cleaved and blastocyst formation rates (86.72 and 86.64; 35.68 and 35.51, respectively, P < 0.05). The highest average total cell number per blastocyst was observed in groups of cells with five and six passages (50.45 and 50.18, respectively). Experiment 2 was performed to assess the sex of donor cells on the subsequent development of SCNT embryos. There was no significant difference in the cleaved and blastocyst formation rates, and the average total cell between female and male groups (86.51 % vs 86.94 % and 35.31 % vs 35.08 %, 50.29 % vs 50.67 %, respectively, P > 0.05). Experiment 3 was performed to assess the effect of cell lines on the development of SCNT embryos. Our results showed no significant difference in the success rate of fibroblast nuclear transfer into recipient oocytes, the cleaved and blastocyst formation rates, and the average total cell number per blastocyst among the cell lines 6004, 9154, 9155, 9156 and 9157 (P > 0.05). Experiment 4 was performed to assess the ability of SCNT embryos to induce pregnancy and to develop term. SCNT embryos were produced from Ỉ fibroblast cells established based on the results of Experiments 1, 2 and 3. Transfer of blastocyst stage embryos into 19 recipients (100-120 embryos in each) resulted in 14 pregnancies, in which 8 pregnant females terminated on Day 22-42 and 6 others produced 20 cloned piglets from donor cells of a female pig but 5 piglets died before birth and 15 healthy cloned piglets. However, 3 out of 15 healthy piglets died of unknown causes within 24h of birth and 3 out of 15 healthy piglets died at 3-5 days of age due to diarrhoea, 9 out of 15 healthy piglets are now 3 months of age. Finally, we established a protocol for the donor cell production which enabled the production of the endangered Ỉ pig embryos by SCNT and maximized blastocyst production rate by more than 35 % and pregnant rate after the transfer of cloned Ỉ pig embryos to recipients at 73.68 % for the first time in Vietnam.
我们的目的是通过体细胞核移植（SCNT）来建立有效的供体细胞，以生产濒危的越南伊比利亚猪的猪仔。在实验 1 中，我们评估了细胞传代对 SCNT 胚胎体外发育的影响。具有五和六传代的细胞表现出明显的分裂和囊胚形成率（分别为 86.72%和 86.64%；35.68%和 35.51%，P<0.05）。在五和六传代的细胞组中，每个囊胚的平均总细胞数最高（分别为 50.45 和 50.18）。实验 2 是为了评估供体细胞的性别对 SCNT 胚胎随后发育的影响。雌性和雄性组之间的分裂和囊胚形成率以及每个囊胚的平均总细胞数没有显著差异（分别为 86.51%比 86.94%和 35.31%比 35.08%，50.29%比 50.67%，P>0.05）。实验 3 是为了评估细胞系对 SCNT 胚胎发育的影响。我们的结果表明，在纤维母细胞核转移到受体卵母细胞的成功率、分裂和囊胚形成率以及细胞系 6004、9154、9155、9156 和 9157 中的每个囊胚的平均总细胞数方面，没有显著差异（P>0.05）。实验 4 是为了评估 SCNT 胚胎诱导妊娠和足月发育的能力。根据实验 1、2 和 3 的结果，从伊比利亚纤维母细胞中产生了 SCNT 胚胎。将囊胚期胚胎转移到 19 个受体中（每个受体中有 100-120 个胚胎），导致 14 例妊娠，其中 8 例妊娠雌性在第 22-42 天终止妊娠，另外 6 例妊娠雌性从雌性猪的供体细胞中产生了 20 头克隆猪，但有 5 头仔猪在出生前死亡，有 15 头健康的克隆仔猪。然而，15 头健康仔猪中有 3 头在出生后 24 小时内死于不明原因，15 头健康仔猪中有 3 头在 3-5 天龄时死于腹泻，9 头健康仔猪现在 3 个月大。最后，我们建立了一种供体细胞生产方案，使通过 SCNT 生产濒危的伊比利亚猪胚胎成为可能，并将囊胚的产生率提高了 35%以上，并使克隆伊比利亚猪胚胎转移到受体后的妊娠率达到 73.68%，这在越南是首次。
