RNA Isolation from Girolando (Bos taurus × Bos indicus) Sperm in Fresh and Cryopreserved Semen for RNA-Seq Applications

The highly compact chromatin and naturally fragmented RNA content of bovine sperm make it difficult to obtain sufficient total RNA for transcriptome sequencing. Bovine sperm studies have mostly adapted the somatic-cell protocol and used generic kits. To date, no commercial extraction kit is available for total RNA from sperm. These limitations prompted optimization of a sperm total RNA isolation protocol, tested using both fresh and cryopreserved sperm. Ejaculates were collected from two Girolando bulls (Bos taurus × Bos indicus) and were subsequently processed for cryopreservation. In total, eight RNA extraction protocols were tested, namely four TRIzol®-based (GP) protocols and four spin-column (SC) methods. Across both fresh and cryopreserved sperm, Protocol D of SC protocols (SC-D) was the most suitable choice for total RNA sequencing. For fresh sperm, ~25–29 million filtered reads were obtained, and cryopreserved sperm yielded ~83–125 million filtered reads, indicating that cryopreserved sperm is also a good source of sperm RNA based on initial sequencing metrics. By pinpointing SC-D as the optimal balance in terms of yield and sequencing performance, and by identifying practical solutions for DNA carryover and storage-related duplication, the study’s results provide a streamlined protocol and quality control framework for total RNA sequencing of Girolando sperm.