Production of Nuclear Transfer Llama (Lama glama) Embryos from In Vitro Matured Llama Oocytes

Abstract

To date, there have been no reports of somatic cell nuclear transfer in llamas. The application of this methodology to the camelid industry could be helpful in the propagation of genetically valuable animals. The objective of this study was to produce nuclear transfer llama embryos comparing the development of these llama embryos cultured in either CR1aa medium (treatment A) or G1.2 medium (treatment B) medium. Llamas were superstimulated by double dominant follicle reduction 12 days apart, followed by pFSH administered in daily descending doses over a 3-day interval (total dose of 200 mg). Animals were ovariectomized by flank laparotomy, follicles were aspirated from excised ovaries and oocytes were in vitro matured for a 30-h period. Adult female llama fibroblasts were used as donor karyoplasts and injected into enucleated llama oocytes. Embryo development was assessed after 2 days of culture. A total of 307 follicles were aspirated from nine treated females, resulting in 298 (97%) oocytes recovered. Of a total of 229 evaluated oocytes, 120 (52%) achieved nuclear maturation. Of a total of 80 reconstructed couplets, 50 (62.5%) were successfully fused. Subsequent cleavage rates were 32 and 40% for treatments A and B, respectively, with no significant difference (p < 0.05) detected between treatment groups. A total of 11 embryos (8-cell to morula stages) were transferred to synchronized recipient llamas. Ultrasonography at 14 days post-transfer indicated that no pregnancies were established. This study shows that nuclear transfer can be successfully applied to the production of llama embryos. Further research is needed to identify optimal parameters to improve efficiency of nuclear transfer in this species.

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

“…This may be explained by the finding of Torner et al (2003) who reported that the oocytes of pregnant camels require 36 h of maturation to reach levels of >50% M)) in comparison with the oocytes of non-pregnant camels, where 32 h are sufficient. Regardless serum supplementation in a maturation medium, in vitro maturation of oocytes depends on several conditions including reproductive status, ages, side of the ovary (Amer and Moosa 2009) and method of oocytes collection (Sansinena et al 2003, Farag et al 2012. The last authors found that the ovaries without the corpus luteum are promising subjects for high recovery value of camel oocytes (COCs) which have a great ability to be matured in vitro.…”

Cloning in companion animal, non-domestic and endangered species: can the technology become a practical reality?

“…Availability of ovaries from local abattoirs and a great understanding of bovine embryo development in vitro and in vivo have played a major role in the success of SCNT in nondomestic bovids. SCNT embryos develop to the blastocyst stage at reasonable rates ranging from 12 to 13% in the gaur (Lanza et al 2000;Mastromonaco et al 2007) to 34% in the buffalo (Saikhun et al 2002); whereas llama SCNT embryos were not capable of developing past the early cleavage stages (Sansinena et al 2003). Both wild cattle species in which transfer of SCNT embryos was attempted resulted in a live birth (gaur: Lanza et al 2000;banteng: Sansinena et al 2005); however, the gaur calf died and one of two banteng calves had to be euthanised within days of birth.…”

“…Nonetheless, it is well documented that interspecies SCNT embryos between highly distinct species exhibit poor developmental potential, usually arresting before activation of the embryonic genome (e.g. rat-domestic cattle: 2-cell stage, Dominko et al 1999; llama-domestic cattle: 8-16-cell stage, Sansinena et al 2003; tiger-domestic cattle: 1.8% blastocyst, Hwang et al 2001; black bear-domestic cattle: 4% blastocyst, Ty et al 2003). According to the results from giant pandaand macaque-rabbit SCNT embryos, if nuclear-mitochondrial communication is established with the replication of the donor mtDNA, then the decreased development exhibited by interspecific embryos must be caused by other cytoplasmic factors.…”

Embryo Preservation and in Vitro Production of Embryos

“…30 These authors used adult male llama fibroblast cell lines obtained from a skin biopsy and IVM oocytes recovered from superstimulated females after ovariectomy. The karyoplasts of a cell from the donor animal are transferred into the cytoplasm of an oocyte from which genetic material has been removed.…”