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

Contents: The aim of this study was to carry out in vitro fertilization using spermatozoa selected with Androcoll-E™ and to evaluate the efficiency of the culture medium DMEM-F12 for in vitro embryo development in the llama. Twelve adult females from 18 superstimulated (67%) were used as oocyte donors. They were superstimulated with 1500IU of eCG and after 5days, received a single dose of buserelin. Twenty hours post-injection, follicular aspiration was conducted by flank laparotomy. Semen collections were performed under general anesthesia by electroejaculation of the male. The ejaculates were processed with a solution of collagenase (0.1%) and an Androcoll-E™ 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 (20×10 6 live spermatozoa/ml) was added to each fertilization microdroplet. After 24h, they were randomly placed in one of two culture media: SOF (n=34) or DMEM-F12 (n=33) and incubated for 6days in humidified atmosphere of 5% CO 2, 5% O 2 and 90% N 2 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-E™ 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. © 2011 Blackwell Verlag GmbH.