Development of prepubertal goat oocytes after their in vitro maturation and chemical activation.

Experiments were conducted to study in vitro maturation of prepubertal goat oocytes and their developmental potential after chemical activation. In Experiment 1, cumulus-oocytes complexes collected from the ovaries of prepubertal goats slaughtered at a local abattoir were matured in vitro in TCM-199-based medium supplemented with 10 µg/ml luteinizing hormone (LH) (treatment 1) or 10 µg/ml LH + 0.1 mM l-cysteine (treatment 2). In Experiment 2, mature oocytes were activated with either 5 µM ionomycin or 7% ethanol. After 18 h, some oocytes were randomly fixed and stained to evaluate their chromatin status, while others were cultured in embryo culture medium to study their further development. In Experiment 3, oocytes activated with 5 µM ionomycin were cultured for 7 days in one of the four different culture media [Charles Rosenkrans medium (CR-1), TCM-199, potassium simplex optimization medium (KSOM) and synthetic oviductal fluid (SOF)] to study their developmental potential. The maturation rate in control, treatment 1, and treatment 2 media did not differ from each other (P > 0.05). However, the lowest degeneration of oocytes was observed in treatment 3 (P < 0.05) when compared with the other two groups. The proportion of activated oocytes was higher, while non-activated oocytes were lower in ionomycin group when compared with the group activated with ethanol (P < 0.05). The proportions of oocytes cleaved were 65.7, 56.8, 61.0 and 54.4% in CR-1, TCM-199, KSOM and SOF medium, respectively, with no significant difference. However, further development of cleaved oocytes was better in KSOM followed by SOF.

Comparison of pregnancy rates with transfer of in vivo produced embryos derived using multiple ovulation and embryo transfer (MOET) with in vitro produced embryos by somatic cell nuclear transfer (SCNT) in the dromedary camel (Camelus dromedaries).

In the present study, there was comparison of pregnancy rates with transfer of in vivo-produced embryos using multiple ovulation and embryo transfer (MOET) with in vitro-produced embryos by somatic cell nuclear transfer (SCNT) in dromedary camels. In vivo-produced embryos were collected from donors after super-stimulation of follicular development on day 7 after ovulation, while in vitro-derived embryos were produced using SCNT from in vivo-matured oocytes collected from camels after follicular development super-stimulation. As a result of estrous synchronization, all recipient camels for both groups were 1 day earlier in stage of estrous cycle than developmental status of embryos at the time of transfer. The animals into which embryos were transferred were monitored at 7-day intervals after embryo transfer for signs of pregnancy based on response to presence of a male and there was ultrasonic confirmation on days 35 and 60 subsequent to day of estrus in recipient animals. A greater proportion of recipients (P < 0.05) were considered pregnant based on response to male presence when there was transfer of MOET-(76.8 ± 3.2) compared with SCNT- (26.4 ± 2.4) derived embryos on day 14. There was no difference in pregnancy losses in subsequent weeks until day 60 between groups. There were also no differences in calving rates of females in which MOET- (91.7%) and SCNT- (93.3%) derived embryos were transferred. These results indicate pregnancies at day 60 with SCNT-derived embryos are sustained for the remainder of gestation periods similar to when there was transfer of MOET-derived embryos in dromedary camels.

Cytoplast source influences development of somatic cell nuclear transfer (SCNT) embryos in vitro but not their development to term after transfer to synchronized recipients in dromedary camels (Camelus dromedarius).

Studies were conducted to evaluate the adequate time for exposure of donor nucleus to oocyte cytoplast before its activation and the effect of oocyte source on the development of SCNT embryos in camels. A higher number of embryos cleaved and developed to blastocyst stage (P < 0.05) when couplets were activated between 1 and 2 h-than that of those activated at 0.5 h or more than 2 h post-fusion. A reduced number of reconstructed embryos cleaved (55.2 ±â€¯7.6%) and developed to the blastocyst stage (20.5 ±â€¯5.5%) when in vitro matured oocytes collected from the slaughterhouse were used as donor cytoplasts, compared to in vitro (71.3 ±â€¯1.3 and 36.7 ±â€¯7.3%) or in vivo matured (91.7 ±â€¯8.3 and 35.4 ±â€¯6.0%) oocytes obtained from live animals (P < 0.05), respectively. However, no differences were observed between the different types of oocyte sources on the establishment of pregnancies and delivery of offspring's. In conclusion, couplets activated 1-2 h post-fusion had higher in vitro developmental potential and oocytes collected from live animals were better in supporting the cleavage and blastocyst production in vitro than oocytes collected from slaughterhouse ovaries, however, all sources of oocytes can be utilized as donor cytoplasts and have the potential to support development of full-term calves after transfer into synchronized recipients.

First cloned Bactrian camel (Camelus bactrianus) calf produced by interspecies somatic cell nuclear transfer: A step towards preserving the critically endangered wild Bactrian camels.

Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies somatic cell nuclear transfer (iSCNT) embryos using skin fibroblast cells of an adult Bactrian camel (Camelus bactrianus) and Llama (Llama glama) as donor nuclei. Also, the embryos reconstructed with Bactrian cells were transferred into the uterus of synchronized dromedary camel recipients to explore the possibility of using them as surrogate mothers. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated mature oocytes, collected from super-stimulated dromedary camels, and fused using an Eppendorf electroporator. After activation with 5µM ionomycin and 6-dimethylaminopurine, they were cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. In experiment 1, Day 7 blastocysts were stained with Hoechst to count their cell numbers, while in experiment 2, they were transferred to synchronized dromedary recipients. A lower number (P < 0.05) of blastocysts were obtained from reconstructs utilizing fibroblast cells from Llama when compared with those reconstructed with dromedary and Bactrian fibroblast cells. However, no difference was observed in their cell numbers. In experiment 2, a higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with Bactrian fibroblast cells when compared to those reconstructed with dromedary cells. Twenty-six Day 7 blastocysts reconstructed with Bactrian cells were transferred to 23 synchronized dromedary recipients with 5 pregnancies established on Day 30, however, only one of the pregnancies developed to term and a healthy calf weighing 33 kgs was born after completing 392 days of gestation. Unfortunately, the calf died on day 7 due to acute septicemia. In conclusion, the present study reports, for the first time, birth of a cloned Bactrian calf by iSCNT using dromedary camel as a source for oocytes as well as a surrogate for carrying the pregnancy to term.