Implementation of a method for sperm cryopreservation in sceloporine lizards

Actual loss of lizard biodiversity continues, even with the implementation of conventional conservation programs. An approach including assisted reproductive techniques such as sperm cryopreservation may contribute to the management of endangered species. We developed a method for sperm cryopreservation in sceloporine lizards and compared the response among the studied species. Prior to the mating season, we obtained semen from adult males of Sceloporus aeneus (n = 21), Sceloporus grammicus (n = 20) and Sceloporus torquatus (n = 21) via pressure of the genital papilla. Volume and sperm concentration were measured before semen dilution in a Tris-egg yolk (TEY) medium to evaluate progressive motility, sperm viability, morphology, plasma membrane and acrosome integrity. Then, we cooled the remaining volumes to 5°C at a rate of 0.1°C per minute to incorporate glycerol (8% v/v) in two fractions. Immediately afterwards, we placed 40 μl of the mix on solid CO₂ to form pellets and immersed them in liquid nitrogen for storage. We thawed the pellets at 29°C for 3 minutes and diluted them 1:1 (v/v) in TEY medium to assess sperm quality. We found a positive relationship between body weight and seminal volume in S. grammicus and S. torquatus and a negative correlation with sperm concentration in S. grammicus (P < 0.05). Moreover, we observed that the freezing-thawing process decreased sperm quality in the three species, mostly affecting motility and viability. However, S. torquatus and S. aeneus showed a higher sperm tolerance than S. grammicus.