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Singina G.N. Change of culture medium positively influences the development and quality of in vitro cattle embryos. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2022, Vol. 57, № 6, p. 1197-1207.
(how to cite this article)

doi: 10.15389/agrobiology.2022.6.1197eng

UDC: 636.2:591.39

Acknowledgements:
Production of IVP embryos was financially supported from the Ministry of Science and Higher Education of the Russian Federation (theme 0445-2021-0004), analysis of embryo quality was financially support from the Russian Science Foundation (project No. 19-16-00115-П).

 

CHANGE OF CULTURE MEDIUM POSITIVELY INFLUENCES THE DEVELOPMENT AND QUALITY OF in vitro CATTLE EMBRYOS

G.N. Singina✉

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

ORCID:
Singina G.N. orcid.org/0000-0003-0198-9757

Received October 17, 2022

Although the practice of producing in vitro embryos (IVP) has become routine in the world, the quality of IVP embryos is still lower than that of in vivo embryos, and the conditions for obtaining in vitro embryos still need to be specified. This original research article presents data on the development and quality parameters of IVP embryos in cattle (Bos taurus), depending on the volume of in vitro culture medium (IVC) and its refreshment protocols. Post mortem bovine oocytes were cultured in a maturation medium, fertilized in vitro with frozen-thawed sperm, and transferred to 500 or 100 µl of BO-IVC medium (IVF Bioscience, UK) for embryonic development. The effects of not refreshing the IVC medium during the 8-day embryonic development period (NMR), half-refreshed medium (HRM) and completely refreshed medium (CRM) were compared. For HRM or CRM, after 3 days of culture, half of the initial 500/100 µl volume was replaced with fresh medium or embryos were transferred to 500/100 µl fresh medium. On day 8 of NMR, HRM, and CRM culture, the number of embryos developed to the blastocyst stage (BL) was evaluated, and the BL quality was assessed as per the total number of nuclei and the apoptotic nuclei counted cytologically. The 8-day-old blastocysts were also cultured for 2 days to the stage of hatched BL (HBL). CRM has been shown to improve embryo production regardless of the volume of the IVC medium when compared to NMR culture. Blastocyst formation increased from 23.0±1.5 and 25.8±0.8 % for 500 µl and 100 µl, respectively, to 45.7±4.8 and 52.1±4.9 % (p < 0.01), while apoptosis decreased from 6.53±0.88 and 6.47±0.66 to 3.60±0.12 and 3.50±0.29 % (p < 0.05 and p < 0.01). The yield of hatched blastocysts increased from 14.9±1.5 and 11.6±3.3 to 25.2±3.9 and 40.8±3.2 % (p < 0.05 and p < 0.001) from the total number of fertilized oocytes. In a 100-µl volume, the number of nuclei in BL also increased from 175.8±13.5 to 224.3±6.7 (p < 0.05). Similarly, the HRM culture was more favorable for embryo development compared to the NMR culture. As in the CRM culture, the blastocyst formation increased up to 44.9±0.7 and 44.1±5.0 % (p < 0.01 and p < 0.01 0.05), and the HBL yield increased up to 26.2±3.2 and 27.7±1.4 % (p < 0.05). In a 100-µl volume, the number of nuclei in BL increased up to 230.4±8.4, (p < 0.05) while the proportion of apoptotic nuclei decreased to 3.10±0.17 (p < 0.01). No significant differences were found between CRM and HRM cultures for all parameters studied. However, with CRM and a 100-µl volume, the rates of BL and HBL were the highest. Thus, in cattle, CRM and HRM cultures, as compared to NRM without medium change, have a positive effect on the development and quality of IVP embryos. The noted effect obviously depends on the volume of the IVC medium. For BO-IVC medium with a volume of 500 µl, both manipulations to refresh the medium are effective, for a volume of 100 µl, CRM is the best.

Keywords: in vitro embryonic development, culture medium, IVP, IVC, cattle.

 

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