112 LLAMA LAMA GLAMA PREGNANCIES FROM VITRIFIED/WARMED BLASTOCYSTS USING A NOVEL COAXIAL CRYOPROTECTANT MICROINJECTION SYSTEM

Abstract

There has been only limited progress in efforts to cryopreserve llama embryos, and no live offspring have been reported to date. The main obstacle is the large diameter of in vivo-recovered llama embryos that results in high embryo volume to surface ratio. First, a novel coaxial microinjection device was designed and constructed to allow the injection of a cryoprotectant (CPA) solution directly into the cavity of hatched llama blastocysts in an effort to reduce the exposure time to toxic CPAs. The coaxial device consisted of a holding borosilicate pipette (120-μm OD; 60-μm ID) and an injection pipette (20-μm OD; 17-μm ID) placed inside the holding pipette for injection of CPAs and for aspiration of aqueous fluids from the blastocele cavity. In this preliminary study, two methods of cryopreservation were evaluated. Treatment (Trt) A consisted of injection of a CPA solution using the coaxial microinjection system followed by a vitrification (VITC) protocol. Day 7 in vivo-derived, hatched llama blastocysts (>500-μm diameter) were held in PBS during injection with an equilibration solution (EQUL) consisting of 15% glycerol, 10% butanediol, 1% polyethylene glycol, 20% fetal bovine serum (FBS) and 0.5 m sucrose in ViGro Holding Plus medium (Bioniche Animal Health, Melleville, Ontario, Canada). Embryos were immobilized with the holding pipette and EQUL solution was injected until the embryos were hyperinflated. The contents of the blastocele cavity were then aspirated and the embryos were released and held in EQUL solution for 8 min. After equilibration, embryos were transferred to a 3 step VITC solution (20% glycerol, 20% ethylene glycol, 0.3 m sucrose, 0.375 m glucose, 3% polyethylene glycol in three steps; Aller et al. 2002 Anim. Reprod. Sci. 73, 121–127), loaded into 0.25-mL plastic straws, and plunged into LN₂. Trt B consisted of VITC only, without microinjection of CPAs. After 60 days, the straws containing the vitrified embryos in Trt A were removed from the LN₂ and placed in a water bath at 32°C for 10 s. Embryos were injected with diluent medium (0.3 m sucrose in ViGro Holding Plus) into the blastocele cavity using the coaxial system until re-inflated to their original spherical shape. The warming of embryos in Trt B was performed without injection of diluent medium. Embryos from both treatments were held in diluent medium for 7 min and then placed in ViGro culture medium. Randomly chosen, VITC-thawed hatched blastocysts were nonsurgically transferred (1 or 2/female) to GnRH-treated llama recipients. Two pregnancies resulted from the transfer of three embryos (67%) from Trt A, with normal heart beats confirmed by ultrasonography 24 days post-transfer (Table 1). No pregnancies resulted from the transfer of embryos from Trt B. The pregnancies remain ongoing (60 and 210 days) at the time of this submission. These preliminary results demonstrate the successful use of a novel microinjection system in the cryopreservation of llama embryos. Research is now underway to determine the optimal VITC solutions and embryo exposure time.

Table 1. Comparison of two vitrification methods for hatched llama blastocysts