METHOD FOR PIG EMBRYONS GENERATION IN VITRO Russian patent published in 2008 - IPC A01K67/02 A61D19/04 

FIELD: biotechnologies.

SUBSTANCE: method includes ovary removal after animal slaughtering, their transportation, ovocytes extraction from ovary, their cultivation to metaphase II stage, preparation of boar sperm, combined cultivation of ovocytes and spermatozoids and cultivation of ovulums. Besides, ovary is separated from animals no later than 30 minutes after the moment of animal immobilisation. Ovary is transplanted in free of antibiotics salt solution at 35-37°C during 1 hour. Ovocytes are taken from ovary by dissectioning of visible antrum-containing follicles. Received ovocytes are split into groups by quantity of surrounding cumuluce cell layers, namely: ovocytes surrounded by 4-5 layers of cumuluce cells, 2-3 layers and one layer of cumuluce layer. Each group of ovocytes is cultivated in medium containing 10 IU of human chorionic gonadotropin. To fertilise ovocytes in vitro, ejaculated sperm of pigs is used, which is dissolved with glucose-chelate-citrate-sulphate diluter and cleaned from semen plasma dissolved with mTBM (mouse thymic virus) medium and incubated during 25 minutes under incubator conditions at 5% CO₂ and 38.5°C. Produced zygotes are cultivated in NCSU-23 medium free of phenol red and enriched with 0.1% of amino acids during 6-7 days; in addition, 0.17 mM Na-pyruvate and 2.75 mM Na-lactate are introduced into NCSU-23 medium during the first three days of cultivation.

EFFECT: increase of mature ovocytes yield and their fertilisation ratio in vitro.

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