Development of a procedure for isolation, identification and quality assessment of bovine spermatids and evaluation of their fertilizing ability in vitro

Rolando Pasquariello¹Francesca Di Filippo¹Sai Kamal Nag Bonumallu²Fernanda Fagali Franchi²Ramona Pistucci³Federica Franciosi²Valentina Lodde²Alessandra Iannuzzi³Alberto Maria Luciano²Tiziana AL Brevini⁴Fulvio Gandolfi^1*

• ¹Department of Agricultural and Environmental Sciences - Production, Landscape, Agroenergy, Università degli Studi di Milan, Milan, Italy
• ²Reproductive and Developmental Biology Laboratory, Department of Veterinary Medicine and Animal Sciences, Università degli Studi di Milano, Lodi, Italy
• ³Institute for the Animal Production System in the Mediterranean Environment, National Research Council (CNR), Naples, Campania, Italy
• ⁴Laboratory of Biomedical Embryology and Tissue Engineering, Department of Veterinary Medicine and Animal Sciences, Università degli Studi di Milano, Lodi, Italy

Intracytoplasmic spermatid injection into oocytes has limited efficiency in cattle, with no offspring generated so far, partly due to ambiguous spermatid identification. This study aimed to develop and validate a method for isolating and characterizing bovine spermatids to improve the efficiency of spermatid intracytoplasmic injection. First, we optimized a protocol for spermatid isolation from bull testis using a discontinuous Percoll gradient and 10 μm mesh cell strainers. Next, we established a stage-specific separation strategy based on DNA content, size, and granularity using flow cytometry to distinguish round and elongating/elongated spermatids suitable for molecular analysis.Morphological assessment confirmed that 72.5% of isolated cells were at the spermatid stage, supported by a high haploidy rate, spermatid-specific transcript expression (PRM1, PRM2, SPACA9, SPERT), and SPERT protein detection. Viability assays showed that spermatids maintained intact DNA at 0 and 24 hours at 4°C and 37°C, though mitochondrial activity and ROS levels increased over time, suggesting oxidative stress. When spermatids were injected into oocytes (n=82), only 13.4% formed two pronuclei, whereas 46.3% exhibited a single pronucleus and a condensed chromatin spot, indicating incomplete activation or fertilization failure. This work contributes to refining bovine intracytoplasmic injection protocols. Future applications of this approach, particularly if functional spermatids can be derived from spermatogonia or embryonic cells, could help shorten the generational interval in cattle breeding.