Avian Cloning: Adaptation of a Technique for Enucleation of the Avian Ovum

The use of reproductive technologies such as somatic cell nuclear transfer (SCNT) for avian species has been limited by the inability to visualise the pronucleus or pronuclei within the blastodisc or germinal disc region, respectively, primarily due to the opacity of the large, lipid filled yolk. The main objective in the present study was to assess a method for visualising and enucleating the avian ovum, a critical step in developing the capability for cloning birds. The method utilised in the present investigation was epi-fluorescence transmitted light (top-side UV) microscopy (EFTLM), also known as fluorescence/oblique or fluorescence/differential interference contrast (DIC) illumination, combined with vital staining and DNA visualisation techniques. The use of EFTLM combined with micromanipulation methods adapted from mammalian cloning procedures showed that the vitelline membrane of the avian ovum can be pierced and aspiration of the pronucleus, once visualised, can be performed without compromising the ovum's structure. Two approaches for domestic chicken ova collection, i.e., in vivo and in vitro, were utilised and ova recovery rates were compared. Based on a statistical analysis, i.e., Fisher's exact test, the results of the in vivo versus in vitro ovulation ova recovery methods were significantly different (P < 0.05), with the in vivo ovulation method yielding more viable intact ova. In conclusion, enucleating the avian ovum using EFTLM combined with vital staining, DNA visualisation, and micromanipulation techniques can be a feasible option for future avian cloning endeavours; although it will require further refinement to improve overall efficiency.