Analysis of sperm processing and cryopreservation procedures in the common marmoset (Callithrix jacchus) to reduce fragmentation and maximize total and progressive motility

Abstract

The marmoset has emerged 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 38C 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.