Kavli Affiliate: Richard Huganir and Xiaoqin Wang
| Authors: Rebecca A Holton, Joshua T Brennan, Sophia I Lipari, James Pickel, Nia Denney, Bhuchitra Singh, Jinwoo Jun, Yang Zhang, Colin D Robertson, Pan P Li, Ingie Hong, Richard L Huganir, Reza Kalhor, Christopher A Ross, James H Segars and Xiaoqin Wang
| Summary:
The marmoset has emaserged as an ideal species for studying complex neurodevelopmental diseases, and IVF is required for gene-editing success. Reliable sperm processing is paramount to several IVF techniques, including traditional insemination. If not used immediately, thermal stress from cryopreservation diminishes sperm quality, requiring improved processing methods. Swim-up and gradient methods may limit adequate sperm quality, and the lack of a standardized freezing protocol often results in impaired post-thaw motility. Thus, reliable standards for sperm processing and cryopreservation in the marmoset are needed to support vital IVF processes. Collected semen underwent one of four pre-processing incubation steps before processing by swim-up, sucrose gradient, or a microfluidics assay. Sperm parameters were assessed using a Hamilton Thorne Computer Assisted Sperm Analysis (CASA) system. To evaluate cryopreservation, microfluidics-processed sperm were subjected to five protocols using different cryoprotectants and cooling methods. After thawing, sperm samples were analyzed with the CASA system and a DNA fragmentation assay. Incubation for 30 minutes at 38°C in TYH media decreased sperm aggregation, and subsequent microfluidics resulted in a higher percentage of motile and progressively motile sperm than swim-up or gradient methods. Our optimized cryopreservation protocol consistently achieved at least 20% progressive motility, a key benchmark for usable marmoset sperm, while previously published or commercial methods were less consistent. The cryopreservation protocol outperformed other published methods, having the least significant change in post-thaw motility without causing DNA damage. These findings establish standardized methods for reliably procuring and storing sperm samples for IVF or husbandry in the common marmoset.