In vitro Embryo Production in Llamas ( Lama glama ) from In vivo Matured Oocytes with Raw Semen Processed with Androcoll‐E using Defined Embryo Culture Media

Abstract

The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-ETM and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females were used as oocyte donors. They were superstimulated with 1500 IU of eCG and after five days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collection was performed under general anesthesia by electroejaculation of the male. The ejaculate was processed with a solution of collagenase (0.1%) and an Androcoll-ETM column was used to improve the sample. Sixty nine COCs were recovered from 79 aspirated follicles (87% recovery). Only expanded COCs were used (n=67); they were randomly placed in groups of 1-5 in Fertil-TALP and the sperm suspension (20 x 106 live spermatozoa/ml) was added to each fertilization microdroplet. After 24 h, they were randomly placed in one of two culture media: SOF (n=34) or DMEM-F12 (n=33) and incubated for 6 days in humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38° C. The blastocyst rate was 20% (7/34) in SOF medium (3 hatched, 2 expanded and 2 early blastocysts) and 15% (5/33) in DMEM medium (all expanded blastocysts). In conclusion, using Androcoll-ETM it is possible to select good quality spermatozoa from llama ejaculates for in vitro fertilization and to produce blastocysts in DMEM-F12 medium. This is also the first time that hatched llama blastocysts have been produced after culture in a defined medium such as SOFaa.