title: Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws.
abstract: Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.
authors.affiliation: Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins 80523, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Hurtt
authors.forename: F
authors.initials: F
authors.lastname: Landim-Alvarenga
authors.forename: G E
authors.initials: GE
authors.lastname: Seidel
authors.forename: E L
authors.initials: EL
authors.lastname: Squires
mesh_terms: D000818:Animals; D002417:Cattle / Q000502:physiology*; D002470:Cell Survival; D019855:Ethylene Glycol; D005260:Female; D005362:Ficoll; D006736:Horses / Q000502:physiology*; D009865:Oocytes; D012996:Solutions; D013395:Sucrose; D014021:Tissue Preservation / Q000379:methods / Q000662:veterinary*
chemical_list: D012996:Solutions; D005362:Ficoll; D013395:Sucrose; D019855:Ethylene Glycol