Optimization of sperm cryopreservation protocols for blue tilapia (Oreochromis aureus)

Abstract

Conservation efforts are implemented worldwide in an attempt to mitigate the decline in biodiversity experienced today. As of 2021, over 3000 species of fish worldwide have been classified as critically endangered, endangered, or vulnerable. Primary contributors include climate change, pollution, habitat loss, overfishing, and invasive species. These issues are not being combated fast enough to save species from extinction. Thus, it is vital to develop complementary approaches to conserve biodiversity. An area of research under development is cryopreservation of fish sperm. Sperm cryopreservation provides an excellent method for species conservation by allowing for long-term storage of genetic material without risk of deterioration; therefore, providing a safeguard against species collapse. Unfortunately, cryopreservation is difficult to standardize across fish species due to its physiological complexity as well as biological differences of spermatozoa across species that influence their tolerance to the freeze/thaw process. Thus, species-specific methods must be developed. Blue tilapia (Oreochromis aureus) are of particular interest due to their importance as a food source for humans, excellent adaptability to being in culture, they are easy to breed, reproduce quickly, and tolerate high stocking densities. However, there is limited research on the cryopreservation of blue tilapia sperm, as the focus often lies on Nile and Mozambique tilapia. Therefore, I investigated an optimized cryopreservation protocol for blue tilapia sperm by testing combinations of three different cryoprotectants: dimethyl sulfoxide (DMSO), methanol (MeOH), and glycerol (Gly), with three different slow cooling rates: -1°C/min, -5°C/min, and -10°C/min. After thawing, viability of the protocols was assessed through spermatozoa motility and swimming kinematics as well as cell membrane damage using Trypan Blue membrane stain and a lactate dehydrogenase (LDH) enzyme assay on seminal plasma. Results indicate that DMSO at -5°C/min yielded consistently better outcomes than other treatments, however, due to limited overall success, further investigation is required to achieve the most effective protocol. Finally, percent motility analysis and Trypan Blue staining provided the most reliable results while LDH activity and swimming kinematics were less conclusive as viability measures.