307 ADDITION OF HIGH CONCENTRATIONS OF HEPARIN DURING IN VITRO FERTILIZATION IMPROVES PERCENTAGES OF CLEAVAGE AND BLASTOCYST FORMATION WHEN BOVINE OOCYTES ARE INSEMINATED IN VITRO WITH SEX-SORTED SPERM

Abstract

The developmental potential of oocytes fertilized in vitro by sexed–sorted frozen–thawed sperm remains low. The aim of this study was to find an optimal concentration of heparin during IVF using frozen–thawed sex-sorted sperm to improve the percentages of fertilization and blastocyst formation. A total of 1708 matured bovine oocytes were randomly allocated among different concentrations of heparin (0, 2.5, 5, 10, 20, and 40 µg mL−1) in Bracket-Olifant medium and co-incubated with non-sexed or sex-sorted sperm for 6 h (Day 0). The sperm (sorted or not) used for IVF was from one bull of proven fertility. Presumptive zygotes then were cultured in CR1aa + 6 mg mL−1 of BSA in 5% O2, 5% CO2, and 90% N2 at 39°C until Day 8. Percentages of cleavage were recorded on Day 2. Data (3 replicates) were analyzed by ANOVA. The results (Table 1) demonstrated that addition of different concentrations of heparin in IVF medium for non-sexed sperm groups did not alter cleavage and blastocyst formation. However, the lowest percentages of cleavage were observed with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups, demonstrating that high concentrations of heparin (20 and 40 µg mL−1) positively affected the percentages of cleavage and blastocyst formation. A greater proportion of zygotes in 20 and 40 µg mL−1 sexed sperm groups developed into grade 1 blastocysts on Day 7, and subsequently formed more blastocysts on Day 8 compared with 0, 2.5, and 5 µg mL−1 of heparin in sexed sperm groups. In conclusion, addition of heparin was not necessary when frozen–thawed non-sexed sperm were used for IVF with this particlar bull. However, addition of higher concentrations of heparin during IVF improved fertilization and blastocyst formation in vitro (40 µg mL−1 of heparin could be used as an alternative concentration for frozen–thawed sex-sorted sperm with this particular bull). The incidence of polyspermy as well as polyploidy in blastocysts is currently being studied. Table 1.Effect of heparin concentrations during IVF on cleavage and blastocyst formation