Label-free chromosome identification in bovine sperm cells using quantitative phase microscopy

Abstract

Sperm sex sorting technology allows dairy farmers to control the sex of their calves, improving herd management, reducing inefficiencies, and enhancing economic viability in large-scale animal reproduction. The most widely used method, flow cytometry, relies on fluorescence-based sorting, but this approach can compromise sperm viability and raise concerns about long-term genetic effects. Additionally, validation of sorted sperm typically involves bright-field microscopy and computer-assisted analysis, which provide only qualitative assessments of motility and morphology. This study introduces a novel, label-free approach using partially spatially coherent light-based quantitative phase microscopy (QPM) to differentiate X- and Y-chromosome-bearing spermatozoa. The technique reconstructs phase maps to quantify differences in optical properties and biological characteristics between X- and Y-sperm, eliminating the need for invasive labeling. We use statistical analysis on X-sorted and unsorted spermatozoa to form a basis for sorting X and Y chromosomes with high purity. Beyond chromosome differentiation, this technique enables precise morphological analysis of bovine sperm, including head length, width, area, ellipticity, elongation, perimeter, roughness, thickness, and volume.