Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification |
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| Institution: | 1. Obstetrics and Gynaecology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy;2. Vita-Salute San Raffaele University, Milan, Italy;3. Reproductive Sciences Laboratory, Obstetrics and Gynaecology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy;1. Departamento de Zoologia, Instituto de Biociências, Universidade Estadual Paulista, Campus Rio Claro, SP, 13506-900, Brazil;2. Department of Biological Sciences, Developmental Integrative Biology Cluster, University of North Texas Denton, TX 76203-5017, USA;3. School of Biosciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom |
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| 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. |
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