30 Supplementation of in vitro culture medium with linoleic acid albumin improves bovine embryo survivability in low-temperature storage at 4°C

Abstract

The objective of this study was to investigate the addition of linoleic acid albumin (LAA) to in vitro culture medium to improve the development of blastocysts with low permeability and fluidity in serum-supplemented medium. Currently, vitrification and slow freezing are performed as cryopreservation methods of bovine embryos. Recently, it was reported that in vivo embryos stored at 4°C for 168 h retained their pregnancy competence. For IVF, embryo development and cryotolerance are highly influenced by the presence of serum in the culture medium. Thus, this study aimed to confirm the survivability of in vitro fertilized embryos cultured in medium supplemented with LAA after low-temperature storage. The cumulus-oocyte complex collected from slaughterhouse-derived ovaries was matured for 20 h in tissue culture medium-199 supplemented with 0.02 AU mL⁻¹ FSH and 5% calf serum (CS). After cumulus-oocyte complexes were incubated with 5 × 10⁶ spermatozoa per mL for 6 h and the presumptive zygotes were removed, their cumulus cells were cultured with CR1aa supplemented with 5% CS (control) or CR1aa supplemented with 0.3 mg mL⁻¹ LAA and 5% CS (LAA group) for 7 or 8 days. The code 1 or 2 blastocysts that developed on Days 7 and 8 were used in this study. The developed blastocysts were equilibrated to the low temperature storage medium (25 mM HEPES buffered tissue culture medium-199 supplemented with 50% fetal calf serum (FCS)) for 20 min and stored in a refrigerator at 4 ± 1°C for 48 or 72 h. After low-temperature storage, the blastocysts were diluted in a buffer (Dulbecco's phosphate-buffered saline) supplemented with 10% FCS for 10 min, washed three times with tissue culture medium-199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol, and cultured in the same medium. Thereafter, blastocyst survival (re-expansion) and hatching were observed for up to 72 h. The data were analysed using the chi-squared test with Yates's correction. The results of 48-h low-temperature storage of blastocysts for the control (n = 55) and LAA (n = 28) groups showed significant differences (P < 0.01) in survival (41.8 and 89.3%, respectively) and hatched rates (20.0 and 89.3%, respectively). The results of 72-h low-temperature storage of blastocysts for the control (n = 13) and LAA (n = 38) groups showed significant differences (P < 0.01) in survival (7.7 and 52.6%, respectively) and hatched rates (7.7 and 52.6%, respectively). These results suggested that supplementation of culture media with LAA improved the survivability of expanded blastocysts after low-temperature storage.