Sexagem de sémen de base imunológica para direcionamento da produção animal.

The present project aims to answer a critical challenge in cuniculture, relevant to other animal production sectors: the ethical and economic burden of unwanted sex offspring. We aim to develop a prototype of the first immunological-based sperm sexing kit for rabbits, potentially applicable to other species that share the same cell surface protein target. Our approach targets ATP11C, a sex-specific transmembrane protein in rabbit semen, identified by our research group. It is encoded by the X chromosome and exhibits distinct staining patterns among rabbit spermatozoa (nearly 50-50% distribution of ATP11C-positive (X-sperm) or negative (Y-sperm) staining in the sperm tail). ATP11C and other sex-specific proteins, identified through bioinformatics and mass spectrometry analysis of rabbit and bovine semen samples, are part of a European patent application (EP24160720), currently pending approval. To sex sperm, we will assess if an ATP11C segment/isoform is unique to X-sperm tails or if ATP11C is not expressed in Y-sperm tails. We will determine its 3D structure to identify common surface epitopes for efficient separation, applicable across species. This information will be used to develop an antibody with high specificity/affinity for these epitopes. Magnetic beads coated with it will bind specifically to X-sperm and, when subjected to a magnetic field, bound cells will remain attached to a column while unpolarized ones will be washed out. Compared to flow cytometry or gender ablation sperm sexing techniques, immunological-based kits are approximately four times less expensive. Additionally, the proposed approach is less invasive, as it targets a cell surface protein instead of relying on DNA staining and laser selection or ablation of a sperm type. Moreover, compared to immunological-based sperm sexing methods that manipulate the motility/fertility of X-/Y-sperm, the present proposal offers key advantages. It provides greater precision and specificity by targeting an antigen specific to the X-sperm, ensuring accurate separation without relying on sperm function variations. Increasing the number of consecutive elution can also enhance accuracy. The proposed method optimizes sample use since positively isolated cells can be used for downstream applications, avoiding compromised function of one sperm type, and offering automation potential for consistent results. The successful implementation of such technology would answer significant concerns, namely about (1) animal welfare, since less valuable animals may be subjected to worse care conditions and/or early culling, (2) environmental impact, due to manure and feed production for animals of inferior economic value, and (3) reduced profitability, since the surplus animals still require feeding, housing, and veterinary care, whose costs may exceed their potential value. A precise sex selection option before insemination would greatly improve efficiency/profitability for farmers while reducing animal welfare concerns and environmental impact by significantly reducing unwanted offspring. Farmers could focus on breeding the sex in higher demand, optimizing their production cycles and resources. The proposed immunological technique is predicted to be more cost-effective, user-friendly, and accessible, making it a viable tool for a wider range of farms compared to flow cytometry or other immunological-based methods, which can be expensive, invasive, technically challenging, or of medium accuracy.