In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: effect of chemical activation treatments and culture conditions.

Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.

Banteng (Bos javanicus) embryos and pregnancies produced by interspecies nuclear transfer.

The banteng (Bos javanicus), a member of the bovidae family, is currently listed as threatened by the IUCN Red List and it is estimated the total world population is <10,000 animals. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer (NT), and an approach such as interspecies NT may be the only alternative to produce embryos and offspring. A total of 348 enucleated domestic bovine oocytes were reconstructed with either male (Treatment A) or female (Treatment B) adult banteng fibroblasts and a total of 103 bovine oocytes were parthenogenically activated as a control (Treatment C). There was no significant difference in fusion rate (68 versus 77%) between Treatments A and B. Of fused couplets, those in Treatment A had greater (P < 0.05) cleavage (67 versus 51%) and blastocyst (28 versus 15%) rate than Treatment B. Of a total of 24 blastocysts transferred into 12 domestic cattle recipients from Treatment A, two pregnancies (17%) were established with heart beats detectable at 30 day by rectal ultrasonography. No pregnancies resulted from the transfer of 14 blastocysts from Treatment B. Both pregnancies were subsequently lost, one between 30 and 60 days and the second between 60 and 90 days of gestation. The bovine cytoplast supported mitotic cleavage of banteng karyoplasts, and was capable of reprogramming the nucleus to achieve blastocyst stage embryos and pregnancies in exotic bovids.