title: Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification.
abstract: The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n = 181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O2. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O2: 62% vs. 5% O2: 25%, P < 0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O2 than under 5% O2 as indicated by lower total and trophectoderm cell numbers (respectively 79 +/- 6 and 56 +/- 6 at 20% O2 vs. 100 +/- 10 and 74 +/- 10 at 5% O2, P < 0.05), by an altered ICM/trophectoderm ratio (20% O2: 28% vs. 5% O2: 23%, P < 0.05), by a higher total nuclear fragmentation (20% O2: 3.7% vs. 5% O2: 1.5%, P < 0.05) and a trend to decreased hatching (20% O2: 32% vs. 5% O2: 81%, P = 0.07). Whereas, for BRL co-culture, 20% O2 yielded higher quality blastocysts than 5% O2 as evaluated by higher ICM and trophectoderm cell numbers (19 +/- 1 and 71 +/- 5 at 20% O2 vs. 15 +/- 2 and 48 +/- 9 at 5% O2, respectively, P < 0.05), by lower nuclear fragmentation in the ICM (20% O2: 2.2% vs. 5% O2: 6.7%, P < 0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O2 seems to be the best combination to evaluate blastocyst survival and quality after vitrification.
authors.affiliation: Unité des Sciences Vétérinaires, UCL, Louvain-La-Neuve, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Kaidi
authors.forename: I
authors.initials: I
authors.lastname: Donnay
authors.forename: A
authors.initials: A
authors.lastname: Van Langendonckt
authors.forename: F
authors.initials: F
authors.lastname: Dessy
authors.forename: A
authors.initials: A
authors.lastname: Massip
mesh_terms: D000818:Animals; D006690:Bisbenzimidazole / Q000737:chemistry; D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D002417:Cattle / Q000196:embryology* / Q000502:physiology; D002460:Cell Line; D018920:Coculture Techniques; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D005456:Fluorescent Dyes / Q000737:chemistry; D006107:Granulosa Cells / Q000166:cytology; D008099:Liver / Q000166:cytology; D008297:Male; D009865:Oocytes / Q000502:physiology; D010100:Oxygen / Q000502:physiology; D011247:Pregnancy; D011419:Propidium / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D011915:Rats, Inbred BUF; D013084:Sperm-Ovum Interactions / Q000502:physiology
chemical_list: D003451:Cryoprotective Agents; D005456:Fluorescent Dyes; D011419:Propidium; D006690:Bisbenzimidazole; D010100:Oxygen