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Singina G.N., Lopukhov A.V., Shedova E.N. In vitro development of cloned embryo in cattle in relation with fusion and activation parameters. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2020, Vol. 55, № 2, p. 295-305.
(how to cite this article)

doi: 10.15389/agrobiology.2020.2.295eng

UDC: 636.2:591.39:576.5

Supported financially by the Russian Foundation for Basic Research (project No. 18-29-07089) and the Ministry of Science and Higher Education of the Russian Federation

 

in vitro DEVELOPMENT OF CLONED EMBRYO IN CATTLE IN RELATION WITH FUSION AND ACTIVATION PARAMETERS

G.N. Singina, A.V. Lopukhov, E.N. Shedova

Ernst Federal Science Center for Animal Husbandry, 60, pos. Dubrovitsy, Podolsk District, Moscow Province, 142132 Russia, e-mail g_singina@mail.ru (✉ corresponding author), shedvek@yandex.ru

ORCID:
Singina G.N. orcid.org/0000-0003-0198-9757
Shedova E.N. orcid.org/0000-0002-9642-2384
Lopukhov A.V. orcid.org/0000-0002-1284-1486

Received December 17, 2019

 

Embryo production through somatic cloning technology has the perspectives for application in reproductive biotechnologies in cattle in order to multiply the most productive and unique genotypes in livestock breeding and create new genotypes using genome editing. Success of somatic cloning depends on the ability of donor somatic cell nucleus (karioplast) to be reprogrammed to totipotent state. Relevant transformations of donor nucleus are mediated by oocyte cytoplasmic factors (cytoplasts) and start from the moment of their association (fusion). Effects of oocyte cytoplasm are direct and depend on many factors. The objective of the present study was to evaluate the cloning efficiency in terms of time of oocyte cytoplasm exposure to donor nucleus before activation, the time of oocyte maturation before their activation in the fused complexes (cytohybrids), and repeated electrofusion of the cytoplast and karyoplast. The effects of these factors on formation of cloned embryos and development to blastocyst stage were studied. Isolated oocyte-cumulus complexes (OCCs) were in vitro matured in TC-199 medium supplemented with 10 % fetal bovine serum, 10 μg/ml of FSH and 10 μg/ml of LH. After 20-24 h of maturation, OCCs were treated with a 0.1 % hyaluronidase, then cumulus cells were mechanically removed and the oocytes with the first polar body were selected. Long-time conserved fetal fibroblasts were in vitro cultured up to monolayer and maintained in contact inhibition during 2 days. Then, cell suspension was prepared for transferring into enucleated oocyte. Somatic cell was transferred to perivitelline space of the oocyte, and two consecutive rectangular 20 μs pulses at constant current with a voltage of 35 V were performed (once or twice if there were no signs of cell-oocyte fusion). The obtained cytohybrids were activated with the ionomycin 1 or 2 hours after fusion (recipient oocytes were matured either 23-25 hours or 26-28 hours). Activated cytohybrides were then cultured up to blastocyst stage. Oocyte cleavage rate were similar in all experimental groups (60.7 to 70.4 %). Blastocyst development rate did not differ between the groups where single or double fusions were performed (29.4±4.4 and 22.8±3.5 %, respectively). Blastocyst rate was 17.4±2.6 % at 1-hour interval between fusion and activation. Two-hour interval increased blastocyst rate to 31.1±3.8 % (p < 0.05). In the case of early activation (23-25 hours of maturation), 29.4±4.8 % of fused complexes developed to the blastocyst stage. With an increase of oocyte maturation time to 26-28 hours, blastocyst rate decreased to 14.6±2.2 % (p < 0.05). Therefore, cloning efficiency depends on the interval between cytohybrid fusion and activation, and the age of MII oocytes at the time of activation of the fused complexes; 2 hours and 23-25 hours, respectively, were the optimal parameters. In addition, the repeated electrofusion of the enucleated oocytes and somatic cells did not affect cytohybrid quality, and, therefore, this procedure can be used for somatic embryo cloning in cattle.

Keywords: cattle, somatic cell nuclear transfer, fusion, activation, embryo development.

 

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