7 Pregnancy from a vitrified-warmed alpaca pre-implantation embryo

The alpaca (Vicugna pacos) is a ruminant livestock species in the South American camelid family. There are more than 9 million South American camelids globally that make important contributions to the livelihoods of rural farmers through conversion of low quality roughages to high quality food and fibre. Reproductive biotechnologies for alpacas are not well developed compared with those for other ruminant livestock species. In particular, embryo cryopreservation technologies are lacking. The objective of this study was to evaluate under field conditions a vitrification protocol originally developed for old world camels that we adapted for use in alpacas. Potential donors were evaluated for follicular development using a 7.5-MHz ultrasound probe. Hembras (sexually mature female alpacas) with ovarian follicles 7-10mm in diameter were behaviour tested to determine sexual receptivity, and receptive females were naturally mated to a proven herd sire. At the time of breeding, non-superovulated donors (n=4) received 30μg gonadorelin. Embryos were nonsurgically collected 7 days after breeding and handled at 20°C. Diameter of harvested embryos (n=4 quality grade 1 hatched expanded blastocysts) was measured using an eyepiece reticle. All recovered embryos were placed individually into 0.5-mL drops of vitrification solution (VS1: 1.4M glycerol) for 5min, 0.5-mL drops of VS2 (1.4 M glycerol + 3.6M ethylene glycol) for 5min, 0.05-mL drops of VS3 (3.4 M glycerol + 4.6M ethylene glycol) for 20s, and 0.05-mL drops of VS3 for 20s while loading into open-pulled straws (OPS). Each OPS was plunged directly into liquid nitrogen for storage for 29 days. At warming, each OPS was submerged into a 1-mL drop of warming solution 1 (WS1: 0.5M galactose) for 1min followed by 1min in WS2 (0.25 M galactose) for 5min before being incubated at 37°C in 5% CO2 in humidified air for 21h in 1mL of Syngro holding medium supplemented with 10% (vol/vol) alpaca serum. Embryos that grew during culture (n=2) were transferred individually into synchronous recipients, and embryos that did not appear to grow (n=2) were transferred together as a pair. Prior to embryo transfer, potential recipients were evaluated ultrasonographically as described previously. Hembras with ovarian follicles 7-10mm in diameter were behaviour tested, and sexually receptive females received 30μg gonadorelin 6 days before embryo transfer. Final selection of recipients (n=3) was based on presence of a corpus luteum and nonreceptive behaviour to a herd sire 24h before transfer. Pregnancy was detected ultrasonographically, and fetal heartbeat was detected 29 days post-transfer in one of the three recipients. Ultrasound at 177 days post-transfer revealed that the pregnancy, generated from a 400μm×375μm vitrified-warmed embryo that had grown in culture, was still ongoing. If this pregnancy results in the birth of a live cria (newborn alpaca), it would represent-to the best of our knowledge-the world's first cria born from a cryopreserved alpaca pre-implantation embryo. It would also demonstrate the potential utility of this protocol under field conditions.