(379) Development of a Consistently Reliable Method for the Processing and Cryopreservation of Common Marmoset

Joshua Brennan1*, Rebecca A. Holton1*, James Pickel1, Bhuchitra Singh1, Nia Bryan2, Xiaoqin Wang2, James Segars1 

1 Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 2Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA 

* Authors contributed equally to this research. 


Abstract Text:

The marmoset has emerged as an ideal species for study of complex neurodevelopmental diseases, and IVF is required for gene-editing success. Reliable sperm processing methods are paramount to several IVF techniques, including intracytoplasmic sperm injection (ICSI), intrauterine insemination (IUI), and fertilization of eggs. If not used immediately, sperm can be cryopreserved, but thermal stress diminishes sperm quality, requiring improved processing methods. Currently reported swim-up and gradient methods in the marmoset may not provide adequate sperm quality, and the lack of a standardized freezing protocol often results in impaired post-thaw motility, requiring more frequent collections with less viable samples. Thus, there is a need to establish a reliable method for sperm cryopreservation in the marmoset to support vital IVF processes. Semen was collected with Ferticare 2.0 vibrator and immediately added to microfuge tubes containing 950 µL warmed and equilibrated (pre-gassed) modified Krebs-Ringer bicarbonate TYH medium. After liquifying in TYH for 30 minutes at 38°C, sperm was processed using either the gradient method, the ZyMot method, or a swim up method. To freeze, sperm processed via the ZyMot method were subjected to 5 different protocols as follows: Origio Sperm Freezing Medium, the O’Brien 2003 protocol, the Morrell 1998 protocol, the Ogonuki 2018 protocol, and a protocol adapted from the NIH. Sperm were then thawed as described in each respective protocol. A Hamilton Thorne CASA system was used to analyze the sperm parameters. Data collected were analyzed using the Student’s T Test, paired or unpaired, as appropriate. When compared to published means of sperm processing, including the gradient and swim up methods, we found that ZyMot resulted in a higher percentage of both motile sperm and progressively motile sperm. When compared to initial sperm parameters after collection, motility in the ZyMot samples was significantly improved (total motile sperm p=0.002, progressively motile sperm p=0.0184). ZyMot was the only processing technique that resulted in an increase in the percentage of motile sperm (means of ZyMot, gradient, and swim up, respectively, were 91.43%, 53.3%, 48.25%; compared to an initial mean of 71.85%). The sperm cryopreservation protocol consistently led to a progressive motility of 30% or higher, a key benchmark for usable sperm in marmosets. Additionally, the use of a small vial for a test thaw allowed us to identify suitable samples prior to thawing on day of use, resulting in fewer thaws for IUI or IVF insemination. This sperm cryopreservation protocol outperformed other published methods, having the least significant change in post-thaw motility (total motility p=0.008696, progressive motility p=0.0494), and the smallest mean percent motility of all methods tested (53.37%, compared to between 72.27-91.53 for other methods). The method offers a reliable means of procuring and storing sperm samples for uses at IVF or husbandry, or other uses in marmosets. Supported by U01DA056556.