Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability.
To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.
Diez
C
C
INRA, Unité de Biologié du Développement, Jouy en Josas, France. carmendm@princast.es
Heyman
Y
Y
Le Bourhis
D
D
Guyader-Joly
C
C
Degrouard
J
J
Renard
J P
JP
D000818:Animals; D001755:Blastocyst / Q000737:chemistry / Q000502:physiology; D002417:Cattle / Q000196:embryology*; D002522:Chlorocebus aethiops; D018920:Coculture Techniques; D015925:Cryopreservation; D003470:Culture Media; D046508:Culture Techniques; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D005312:Fetal Blood; D008055:Lipids / Q000032:analysis*; D008854:Microscopy, Electron; D011247:Pregnancy; D014709:Vero Cells; D015053:Zygote / Q000254:growth & development* / Q000502:physiology
D003470:Culture Media; D008055:Lipids