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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.

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Summary 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.

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The objective of the study was to examine the effect of fetal calf serum (FCS) and mid estrus cow serum (ECS) in culture media on in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes from crossbred cattle in the tropics. Oocytes from abattoir ovaries were cultured in three different maturation media at 39oC temperature, 5% CO2 tension with maximum humidity for 24 h. TCM-199 containing 25mM HEPES, 1mM glutamine L, 2.2mg/mL sodium bicarbonate, antibiotics, 22 µg/mL pyruvate, 1µg/mL estradiol-17β, 0.5µg/mL FSH and 0.06 IU hCG without serum supplement used as treatment-1 and the above media were further supplemented with 10% FCS as treatment-2. In treatment-3, instead of 10% FCS, 20% heat inactivated ECS (serum collected in mid estrum) was used as serum supplement. Oocytes with maximum degree of cumulus expansion were selected as matured and used for further IVF studies using frozen semen. The IVF medium consisted of Fert-TALP medium supplemented with 1µM epinephrine, 10µM hypotaurine, 20µM pencillamine and 0.56µg/ml heparin. Culture conditions set for IVF were 39oC temperature, 5% CO2 tension with maximum humidity. Oocytes showing sperm penetration evidence like presence of enlarged sperm head, male pronuclei with its accompanying sperm tail in the cytoplasm, oocytes with two pronuclei and a clear second polar body but without a sperm tail were considered as fertilized. Significantly higher result of cumulus expansion percentage (p< 0.05) was observed when oocytes matured in media supplemented with FCS as compared to other two treatments. There was no influence of source of serum in maturation media on further IVF of matured oocytes in this study. Int J Appl Sci Biotechnol, Vol 4(3): 308-310

The present study was conducted in dromedary camels to evaluate the effect of the body condition of donor animals on the outcome of superstimulation and embryo collection in experiment 1 and the establishment of pregnancies in the recipients in experiment 2. The animals were physically evaluated for their body condition and scored on a scale of 0 to 5 (0 being very thin and weak, while 5 being very fat). They were divided into three groups based on their body condition score (BCS). Animals with a score of 1–2, &gt;2–3, and &gt;3–4 were divided into groups 1, 2, and 3, respectively. Animals with a score of &lt;1 and &gt;4 were considered either too weak or too fat and were not included in the study. In Experiment 1, 32 donors in 3 groups were superstimulated with a combination of 2500IU of equine chorionic gonadotrophin (eCG, Folligon; Intervet), given as a single intramuscular injection on Day 1 of the treatment protocol, and 400mg of pFSH (Folltropin; Bioniche) injected twice daily in declining doses of 2×80mg, 2×60mg, 2×40mg, and 2×20mg over 4 days, also beginning on Day 1. The ovaries of all the donor camels were scanned on Day 4 after the start of treatment, and thereafter at intervals of one or two days until the majority of follicles had grown to between 1.3 and 1.8cm in diameter before mating with a fertile male. Each donor received an intramuscular injection of 20µg of gonadotrophin-releasing hormone (Receptal, Intervet) immediately after mating, and embryos were collected by non-surgical method on Day 7 after ovulation. In Experiment 2, 141 recipients divided in 3 groups, as described above, were synchronized with donors and received an embryo each in their left uterine horn. The pregnancy confirmation was done by an ultrasound examination on Day 50 after transfer. Data were analysed by ANOVA with Fisher protected least significant difference test (Minitab statistical software, Minitab Ltd.). In experiment 1, the mean±s.e.m. number of follicles (13.9±1.3; 12.7±2.9; 15.0±1.7), and the percent mean±s.e.m. of embryos collected (41.5±7.7; 57.0±12.3; 60.4±7.8) were not different among groups 1, 2 and 3, respectively. However, the percent mean±s.e.m. of transferrable embryos obtained were higher (P&lt;0.05) in group 3 (77.7±4.8) compared with group 1 (57.5±10.1). Similarly, in experiment 2, the percent mean±s.e.m. of pregnancies achieved in group 3 (68.9±6.4) was similar to that of group 2 (57.9±14) but higher (P&lt;0.05) than that of group 1 (50.5±10.0). In conclusion, to the best of our knowledge, this is the first study showing a direct correlation between the BCS and outcome of embryo transfer program in camels. Donors with a good BCS produced more transferrable embryos, and conception rates were higher in recipients having a better BCS. This clearly shows that not only is the selection of recipients critical in the embryo transfer program, but donors with a positive energy balance yield better transferrable embryos. Priming of donors before the start of the breeding season could help to improve the outcome of this technology in camels.