In vitro production of viable bovine blastocysts in media supplemented with rabbit-originated products

Abstract

This study aimed at producing viable bovine blastocysts by IVM, IVF and IVC, in media without addition of bovine-originated products. Bovine serum albumin (BSA) was replaced by polyvinyl alcohol in washing media, fetal calf serum (FCS) by rabbit serum during maturation and culture, BSA by rabbit serum albumin in fertilization and culture media. The effect of substituting rabbit protein sources for bovine ones was tested on nuclear maturation, fertilization and developmental rates, and on the quality of the blastocysts obtained. The proportion of oocytes reaching metaphase II (MII) after maturation in TCM 199 containing epidermal growth factor was similar when supplemented with FCS (90/96) or with rabbit serum (92/99). The rate of 2 pronuclei formation was similar after sperm preparation and fertilization in Tyrode's medium supplemented with BSA (70/97) or rabbit albumin (70/94). In a third set of experiments, a total of 792 oocytes was matured, fertilized and cultured in media supplemented with bovine (standard) or rabbit products. Embryos were co-cultured with their own cumulus cells in synthetic oviduct fluid medium supplemented with serum at Day 3 post insemination. The proportion of cleaved and 5 to 8-cell stage embryos at Day 3 and the blastocyst yield on Day 8 were similar under standard and bovine additive-free conditions (cleavage: 81 vs 80 %, 5 to 8-cell stage: 49 vs 44%, blastocysts: 37 versus 30%). Blastocysts produced under bovine additive-free conditions were viable since a calf was born after the transfer of a frozen embryo. Replacement of bovine products by rabbit originating ones resulted in a 0.5-d delay in blastocyst detection. Day 8 blastocysts produced in media containing rabbit products had a significantly lower number of cells (101 ± 7) than those produced under standard conditions (120 ± 6), possibly because of the difference in blastocyst age. In conclusion, the protocol presented here allows for the efficient production of viable bovine blastocysts under conditions that reduce the risk of disease transmission.