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Singina G.N., Lopukhov A.V., Shedova E.N., Zhukova A.S. The influence of oocyte and do-nor cell preparation conditions on the efficiency of somatic cloning in sheep (Ovis aries L.). Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2024, Vol. 59, № 4, p. 692-703.
(how to cite this article)

doi: 10.15389/agrobiology.2024.4.692eng

UDC: 636.32/.38:591.39:576.5

Acknowledgements:
To conduct the research, the equipment of the Center for Shared Use "Bioresources and Bioengineering of Farm Animals" of the Ernst Federal Research Center for Animal Husbandry was used.
Supported financially by the Russian Science Foundation (grant No. 21-66-00007)

 

THE INFLUENCE OF OOCYTE AND DONOR CELL PREPARATION CONDITIONS ON THE EFFICIENCY OF SOMATIC CLONING IN SHEEP (Ovis aries L.)

G.N. Singina ✉, A.V. Lopukhov, E.N. Shedova, A.S. Zhukova

Ernst Federal Research Center for Animal Husbandry, 60, pos. Dubrovitsy, Podolsk District, Moscow Province, 142132 Russia, e-mail g_singina@mail.ru (✉ corresponding author), vubi_myaso@mail.ru,  shedvek@yandex.ru,  anastasia.s.belyaeva@gmail.com

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
Zhukova A.S. orcid.org/0000-0003-1155-014X

Final revision received June 05, 2024

Accepted February July 08, 2024

 

Obtaining embryos through somatic cell nuclear transfer (SCNT) is a reproductive biotechnology that can be applied in sheep farming to address issues related to the reproduction and preservation of valuable animals, as well as the creation of new genotypes through genome editing methods. However, the efficiency of cloning technology in Ovis aries remains comparatively low, necessitating the optimization of its various stages. In this context, the objective of this study was to assess the efficiency of SCNT based on the in vitro maturation (IVM) environment of oocytes, their age at the time of nuclear transfer (NT,  procedure which includes enucleation of oocytes followed by transfer somatic cell into the perivitelline space of the obtained cytoplasts), as well as the preparation conditions of the somatic cells (duration of serum starvation in culture and storage time in suspension before NT). The impact of these factors on the formation of cytohybrids during the fusion of enucleated oocytes and somatic cells (fusion rate), as well as on the development of cloned embryos (cleavage rate of cytohybrids), was studied. Post mortem cumulus-oocyte complexes (COCs) were matured in TC-199 or in BO-IVM medium from IVF Bioscience (United Kingdom). Oocytes with the first polar body (PB1), aged between 20 to 23 hours (from the start of IVM), were used for NT, along with fetal fibroblasts (FFBs) as donor somatic cells. The latter were cultured to 80-100 % confluence, followed by incubation under serum starvation conditions (to synchronize the cell cycle) for 24 or 48 hours. FFBs were prepared for transfer into the enucleated oocyte in suspension, stored until NT for various durations: ≤ 30, 31-90, 91-150, and > 151 minutes. The cell complexes resulting from NT reconstruction underwent electrofusion, obtained cytohybrids were activated, and then cultured for 2 days for embryonic development. The fusion rate did not significantly differ between experimental groups, ranging from 31 to 42 %. The cleavage rate of cytohybrids also did not differ when oocyte maturation occurred in TCM-199 or BO-IVM medium (47.1±2.40 % and 50.9±3.30 %, respectively). When NT was performed using 22-hour-old oocytes, the yield of cloned embryos was 55.8±3.78 %. Earlier-stage oocytes showed comparable results, while older oocytes (23 hours) significantly reduced the yield (to 39.3±6.69 %, p < 0.05). Extending the serum starvation period of FFBs from 24 to 48 hours also increased the cytohybrid cleavage rate (45.0±4.21 vs. 55.4±4.50, p < 0.05). The duration of FFBs storage did not significantly affect the cleavage rate, but the highest value (56.6 %) was observed with FFBs stored for 31 to 90 minutes, and the lowest when storage time exceeded 150 minutes (40.8 %). Thus, the efficiency of obtaining early-stage cloned embryos in O. aries depends on the age of the oocytes at the time of NT and the duration of FFBs serum starvation before NT. The optimal parameters for the described protocol are 20-22 hours and 48 hours, respectively. Additionally, BO-IVM medium can be used for the maturation of source oocytes when producing cloned sheep embryos. Overall, the identified patterns can be considered preliminary, as the tested factors may have longer-term effects (particularly on the development of cloned embryos to the blastocyst stage and their quality), where more significant differences between the studied variants may become apparent.

Keywords: domestic sheep, cloning, oocytes, in vitro maturation, somatic cells, serum starvation, embryonic development.

 

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