Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer.
This study examined the effects of adding a macromolecule, polyvinylpyrrolidone (10% PVP) and a sugar (0.3 M trehalose) to vitrification solutions (VS) containing either one (40% ethylene glycol [EG], two (25% EG+25% DMSO) or three (20% EG+20% DMSO+10% 1, 3-butanediol [BD]) permeable cryoprotectants on the survival and hatching of IVP bovine embryos, following vitrification, warming and in-straw cryoprotectant dilution. Grade 1 and 2 compact morulae and blastocysts were selected on Day 7 (Day 0=IVF) of culture in SOFaaBSA and equilibrated for 10 min at room temperature in 10% EG. Following exposure, for up to 1 min at 4 degrees C, to one of the above VS (with or without PVP+trehalose), the embryos were loaded into straws and immersed in liquid nitrogen. Following warming and in-straw cryoprotectant dilution, the embryos were cultured for 48 h to assess hatching. There was no effect of VS on the survival of embryos after 24 h, however fewer compact morulae than blastocysts survived after 24 h (24% vs. 75%; P<0.001) or hatched after 48 h (15% vs. 59%; P<0.001). When blastocysts only were considered, an interaction between VS and additional PVP+trehalose was also observed (P<0.01). Hatching was reduced when they were added to 25% EG+25% DMSO (70% vs. 45%) but was not affected for either 40% EG (44 and 49%) or to 20% EG+20% DMSO+10% BD (72 and 72%). Pregnancy rates (Day 90 ultrasound) of recipients that were transferred either two non-vitrified or two vitrified (20% EG+20% DMSO+10% BD) blastocysts, did not differ (3/6 [50%] and 11/20 [55%]). However, significantly (P<0.02) fewer recipients that received compact morulae maintained pregnancy to Day 90 although this was not affected by vitrification (fresh vs. vitrified; 1/5 [20%] vs. 3/18 [17]). These data demonstrate that a VS comprising three cryoprotectants, rather than one, enables more embryos to hatch during post-thaw culture and that the survival, following direct transfer of these vitrified embryos, is not different to non-vitrified embryos.
Pugh
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AgResearch Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand. pugha@agresearch.cri.nz
Tervit
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Niemann
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D000818:Animals; D002072:Butylene Glycols / Q000008:administration & dosage / Q000494:pharmacology; D002417:Cattle / Q000196:embryology* / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000008:administration & dosage / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000008:administration & dosage / Q000494:pharmacology; D005314:Embryonic and Fetal Development / Q000502:physiology*; D004973:Estrus Synchronization; D019855:Ethylene Glycol / Q000008:administration & dosage / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D008297:Male; D009865:Oocytes / Q000502:physiology; D010592:Pharmaceutic Aids / Q000008:administration & dosage / Q000494:pharmacology; D011205:Povidone / Q000008:administration & dosage / Q000494:pharmacology; D011247:Pregnancy; D014199:Trehalose / Q000008:administration & dosage / Q000494:pharmacology; D016216:Ultrasonography, Prenatal / Q000662:veterinary
D002072:Butylene Glycols; D003451:Cryoprotective Agents; D010592:Pharmaceutic Aids; C028491:1,3-butylene glycol; D014199:Trehalose; D019855:Ethylene Glycol; D011205:Povidone; D004121:Dimethyl Sulfoxide