Evaluation of two embryo cryopreservation methods in llama on the in Vivo e in Vitro embryonic survival rates

The aim of the study was to evaluate in llama embryos the effect of two cryopreservation methods on the in vivo and in vitro survival rate. Seventy three hatched blastocysts were recovered by a non-surgical technique at day 6.5 after mating from superstimulated llamas. Receptors were randomly allocated to a control group (n=14), vitrification (n=30) and slow freezing (n=29). On vitrification, embryos were exposed to a vitrification solution (VS) containing 20% Glycerol + 20% Ethylene glycol + 0.5 M Sucrose + 10% fetal calf serum (FCS) + 50 μg/ml gentamicin sulfate, and then plunged into liquid nitrogen in 0.25 ml straws. On the slow freezing, embryos were exposed to phosphate buffer saline (PBS) with 1.5 M Ethylene glycol + 10% FCS + 50 μg/ml gentamicin sulfate, loaded in 0.25 ml straws, and cooled at a rate of 0.12 °C/min to 5 °C. Then, further temperature decrease at 5 °C /min rate, to-20 °C, for 5 min at the mouth of the nitrogen tank; finally straws were plunged into liquid nitrogen. For thawing, two dilution solutions were used composed of two sucrose concentrations: 0.5 M and 0.2 M for slow freezing, and 0.25 M and 0.12 M for vitrification. An in vivo evaluation was performed in all embryos of the control group and in 50% of the experimental groups by direct transfer into previously synchronized female recipients. Pregnancy diagnosis was carried out by transrectal ultrasound evaluation at 20 and 30 days. Pregnancy was in 4/13, 2/12, and 0/11 in recipients from control, vitrification and slow freezing groups respectively, without significant difference. For the in vitro evaluation cryopreservated embryos were cultured in PBS + 20% FCS under atmosphere compose of 5% CO2, 20% O2, and 75% N2 at 39 °C for 1 h, then reexpansion was recorded by morphological characteristics. Embryo reexpansion was 75% (9/12) in vitrified embryos and 57.1% (4/7) in slow freezing embryos, and without significant difference. It was concluded that vitrification could be a suitable method for llama embryo cryopreservation.