102 Feasibility of using manually modified micropipettes for embryo biopsy in dromedary camels

Embryo biopsy has potential applications in embryonic sex determination, paternity identification, and prenatal diagnosis of genetic abnormalities and diseases, but most of the reproduction facilities are not equipped with micro-injector instruments required for the procedure. The current study evaluated the feasibility of using handmade pipettes for the biopsy of in vivo dromedary embryos and the possible effect of the procedure on embryo survival and development. A total of 28 transferable hatched blastocysts were recovered nonsurgically from the uteri of superovulated donors 8.5 days after mating. The size of the embryos was between 250 and 1200 μm. Each embryo was placed in a 50-μL drop of holding medium and kept at room temperature. The biopsy was done by handmade micropipettes prepared as follows. Disposable glass Pasteur pipettes (230 mm long, tip internal diameter ~1 mm) were used. The narrow part of the pipette (~2 cm from the tip) was heated directly on a Bunsen burner along the longitudinal axis and was pulled in the opposite direction once it softened. After locating the inner cell mass, a pipette was applied to immobilize the embryo by making a small penetration on the surface while a second pipette was applied to scratch the surface of the embryo in an attempt to detach very few cells. Punctured embryos (n = 16) and control embryos (n = 7) were cultured in a prewarmed in vitro culture medium at 38°C, 5% CO₂, and 5% O₂ for up to 96 h, and their daily expansion was measured. Five more embryos were biopsied and freshly transferred to recipients on Day 7.5 after induction of ovulation. The biopsied cells were placed on slides and stained with Hoechst, which confirmed the identity of scratched material as cells containing the nucleus. All 16 biopsied embryos continued to grow during culture and expanded at comparable rates to their intact counterparts up to 96 h. The average daily growth of the biopsied embryos did not differ from that of the control group (P > 0.05). Five embryos transferred freshly after the biopsy resulted in four established pregnancies, from which three continued beyond Day 60. In conclusion, this study corroborated the practicality of taking biopsies from in vivo dromedary embryos using a simple affordable technique without utilising microinjection instruments.