Development of in vitro matured, in vitro fertilized domestic cat embryos following cryopreservation, culture and transfer

摘要

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 degreesC in 5% O-2, 5% CO2, 90% N-2 for 24 h. In Experiment 1, after IVM/IVF, on Day 2 (n = 56), Day 4 (n = 48) and Day 5 (n = 42) of IVC, embryos were equilibrated for 10 min at 22 degreesC in HEPES (15 mM) Tyrode's (HeTy) with 1.4 M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 degreesC/min to -6.0 degreesC and held for 10 min. After seeding, cooling resumed at 0.3 degreesC/min to -30 degreesC and after a 10 min hold, straws were plunged into liquid nitrogen (LN2). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/O. 125 M S; 0 PG/O. 125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n = 14; Day 4, n = 26; Day 5, n = 35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2 = 79%, Day 4 = 90%, Day 5 = 98%) and was not different from that of controls (Day 2 = 89%, Day 4 = 88%, Day 5 = 96%). Blastocyst development was similar among days both after cryopreservation (Day 2 = 59%, Day 4 = 54%, Day 5 = 63%) and in controls (Day 2 = 55%, Day 4 = 54%, Day 5 = 58%). Mean (S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2 = 152 +/- 19, Day 4 = 124 +/- 20, Day 5 = 121 +/- 24) than in controls (Day 2 = 141 +/- 25, Day 4 = 169 +/- 21, Day 5 = 172 +/- 19). In Experiment 2, embryos frozen on Day 2 (n = 68), Day 4 (n = 49) or Day 5 (n = 73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean = 12.6 +/- 12.4), 4 (mean = 12.2 +/- 3.7) and 6 (mean = 12.0 +/- 1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.