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-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 1500 IU of eCG and after 5 days, 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 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% 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.

In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals

“…In another species of camel (Lama glama), IVP attained 17% at blastocysts stage (Conde et al 2008). Trasorras et al (2012) found a lower rate of blastocysts reaching 9%.Recently, blastocyst rate ranged between 34-36% using TALP culture medium in the same living species (Trasorras et al 2014). As a result of this, cleavage rate of camel oocytes varies according to the type of culture medium (Khatir et al 2005), the size of follicle (Khatir et al 2007) and co-culture with granulosa or oviductal cells (Khatir et al 2004).…”

Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing

“…Similarly, Trasorras et al. () found increased percentages of positive HOST when using SLC for selecting fresh llama sperm.…”

“…Recent research indicates that SLC‐selected sperm also produce less hydrogen peroxide than controls (Morrell, Lagerqvist, Humblot, & Johannisson, ). Enhanced fertility characteristics have been reported for SLC sperm, and their fertility confirmed for some species (stallion, Morrell, Richter et al., ; bull, Thys et al., ; boar, Sjunnesson, Morrell, & González, ; and llama, Trasorras et al., ) despite the removal of seminal plasma proteins and possibly cholesterol during processing (Kruse, Dutta, & Morrell, ).…”

“…This fact renders it more user‐friendly and less time‐consuming than a traditional density gradient. Single layer centrifugation (SLC) has been successfully used for viable sperm selection in the stallion (Morrell, Johannisson, Dalin, & Rodriguez‐Martinez, ; Morrell, Johannisson, & Rodriguez‐Martinez, ), bull (Morrell, Rodriguez‐Martinez, & Andersson, ; Samardzija et al., ; Thys et al., ), boar (Morrell & Wallgren, ), dog (Dorado, Gálvez, Morrell, Alcaráz, & Hidalgo, ), goat (Jiménez‐Rabadán et al., ), donkey (Ortiz et al., ), brown bear (Álvarez‐Rodríguez et al., ), cat (Chatdarong, Thuwanut, & Morrell, ) and llama (Santa Cruz et al., ; Trasorras et al., ). The SLC‐selected populations of sperm have higher motility and membrane integrity than non‐selected sperm samples (Macías‐García et al., ), and also more morphologically normal and functionally competent sperm, and fewer sperm with damaged DNA.…”

“…While colloid selection has been applied to select good quality sperm from llama ejaculates treated with enzymes (Santa Cruz et al., ; Trasorras et al., ), there is no comparative information on the value of SLC for sperm selection and function in dromedary camels. Considering the challenges presented by the low volume and sperm concentration of the dromedary camel ejaculate, and its excessive viscosity (Skidmore, Morton, & Billah, ), the aim of this study was to determine the efficacy of SLC selection of sperm of this species.…”