98 VITRIFICATION OF ZYGOTES SUPPORTS FULL-TERM DEVELOPMENT IN RABBITS

Abstract

Rabbit can serve as an excellent model for human reproduction and relevant disease study. The objective of the study was to develop an effective procedure to preserve the rabbit genome by vitrifying zygotes. Sexually matured New Zealand White rabbits maintained under a 16 h light:8 h dark cycle were superovulated with a regime of two 0.3-mg, two 0.4-mg, and two 0.7-mg injections of FSH with an interval of 12 h between, followed by an injection of 200 IU hCG and mating. Presumptive zygotes were flushed from the oviducts and collected by midventral lapartomy 18 h after hCG injection. One-cell-stage embryos were assigned to pre-equilibration at 38.5°C in HEPES-buffered TCM199 supplemented with 20% FBS + 7.5% ethylene glycol (EG) (v/v) + 7.5% dimethylsulphoxide (DMSO) (v/v) (dehydration medium) for 3 min, and subsequently to 1.0 ml of vitrification medium (TCM199 supplemented with 20% FBS + 20% EG and 20% DMSO), as described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58), at room temperature for 3 min. Five to six embryos per group were then vitrified in a micro-droplet (approximately 1–2 µL) by directly dropping them into a thin layer of liquid nitrogen on the solid surface that generated a super cold surface for vitrification. Vitrified embryos were sequentially warmed, rehydrated in 20% FBS M199 with different concentrations of sucrose, and washed in 20% FBS M199 for 5 min. Warmed embryos were assigned either for further in vitro culture or for embryo transfer to test corresponding developmental potentials. Dutch rabbits were used as recipients in the procedure of asynchronous ovulation induction (22-h delay to zygote donors) by an intramuscular injection of 15 µg of GnRH per doe. Three or four warmed zygotes were surgically transferred into the recipients on the same day of thawing. Pregnancy was monitored by palpation and/or ultrasound on Days 14–16 post-embryo transfer (ET); C-sections were performed on Day 31 to retrieve full-term developed newborns. Preliminary results showed that a 96% (n = 35) post-warming survival rate was achieved with vitrified rabbit zygotes; subsequent cleavage and blastocyst development were 41.6 and 33.3%, respectively. Transfer of a total of 23 warmed embryos into 6 recipients resulted in 5 pregnancies (83.3%, 5/6). Five live kits (21.7%, 5/23) were delivered. Our study suggests that vitrification of rabbit zygotes is a feasible approach for preserving rabbit genetic material. The establishment of suitable conditions for the vitrification of rabbit zygotes could be useful as a model for vitrification of human 1-celled embryos.

The authors thank the staff of the animal facility at the University of Connecticut for animal care and maintenance. This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.