1. National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China 2. Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou 510642, China 3. Guangdong Wens Foodstuff Group Co., Ltd., Yunfu 527400, China
Enucleation is a crucial procedure for mammalian somatic cell nuclear transfer (SCNT), especially for domestic animal cloning. Oocytes of domestic animals such as pigs and cattle contain dark lipid droplets that hinder localization and removal of the nucleus. Using an oocyte enucleation technique that can obtain a high enucleation rate but has minimal negative effects on the reprogramming potential of oocyte for cloning is beneficial for enhancing the outcome of SCNT. In this study, we compared the pig cloning efficiency resulting from blind aspiration-based (BA-B) enucleation and spindle imaging system-assisted (SIS-A) enucleation, and compared the pig SCNT success rate associated with BA-B enucleation and blind aspiration plus post-enucleation staining-based (BAPPS-B) enucleation. SIS-A enucleation achieved a significantly higher oocyte enucleation success rate and tended to obtain a higher in vivo full term development rate of SCNT embryos than BA-B enucleation. BAPPS-B enucleation also obtained significantly higher in vitro as well as in vivo full term development efficiency of cloned porcine embryos than BA-B enucleation. These data indicate that SIS-A and BAPPS-B enucleation are better approaches for pig SCNT than BA-B enucleation.
. [J]. Frontiers of Agricultural Science and Engineering, 2019, 6(1): 61-65.
Chengcheng ZHAO, Junsong SHI, Rong ZHOU, Ranbiao MAI, Lvhua LUO, Xiaoyan HE, Hongmei JI, Gengyuan CAI, Dewu LIU, Enqin ZHENG, Zhenfang WU, Zicong LI. Effects of enucleation method on in vitro and in vivo development rate of cloned pig embryos. Front. Agr. Sci. Eng. , 2019, 6(1): 61-65.
Number of manipulated/successfully enucleated oocytes
Enucleation rate/%
BA-B
384/319
83.07**
SIS-A
322/306
95.03**
Tab.1
Enucleation method
SCNT embryos
Cleaved/%
Blastocysts/%
Total number of cells per blastocyst
BA-B
240
183 (76.25)
44 (18.33)
40±3.34
SIS-A
279
208 (74.55)
37 (13.26)
47±4.10
Tab.2
Enucleation method
Total number of transferred SCNT embryos
Total number/pregnant/farrowed recipients
Pregnancy/farrowing rate/%
Total number of cloned piglets born
Development rate/%
BA-B
7469
33/23/14
69.70/42.42
64
0.86
SIS-A
6779
33/23/14
69.70/42.42
75
1.11*
Tab.3
Enucleation method
SCNT embryos
Cleaved/%
Blastocysts/%
Total number of cells per blastocyst
BA-B
429
326 (75.99)
94 (21.91)
36±1.77*
BAPPS-B
501
362 (72.26)
89 (17.76)
41±1.69*
Tab.4
Enucleation method
Number of manipulated /successfully enucleated oocytes
Total number of transferred SCNT embryos
Total number/pregnant/farrowed recipients
Pregnancy/farrowing rate/%
Total number of born cloned piglets
Development rate/%
BA-B
8023/not determined
7038
31/18/7
58.06/22.58*
24
0.34**
BAPPS-B
9049/7234
6735
31/21/15
67.74/48.39*
55
0.82**
Tab.5
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