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.

Sperm capacitation involves biochemical and physiological changes that enable sperm to fertilize the oocyte. It can be induced in vitro under controlled conditions that simulate the environment of the oviduct. While extensively studied in mammals, its approach in lizards remains absent. Understanding the mechanisms that ensure reproduction is essential for advancing the implementation of assisted reproductive technologies in this group. We aimed to perform a sperm analysis to determine if capacitation-related changes were induced after incubation with capacitating media. Fifteen males of Sceloporus torquatus were collected during the early stage of the reproductive season. The sperm were isolated from the seminal plasma and then diluted up to a volume of 150 μL using BWW medium to incubate with 5% CO₂ at 30 °C for a maximum duration of 3 h. A fraction was retrieved hourly for ongoing sperm assessment. The sperm analysis included assessments of its motility, viability, the capacitation status using the chlortetracycline (CTC) assay, and the acrosome integrity with the lectin binding assay to detect changes during incubation. We found that total motility was maintained up to 2 h post incubation, after which it decreased. However, sperm viability remained constant. From that moment on, we observed a transition to a deeper and less symmetrical flagellar bending in many spermatozoa. The CTC assay indicated a reduction in the percentage of sperm showing the full (F) pattern and an increase in those exhibiting the capacitated (B) and reactive (RA) patterns, accompanied by an elevation in the percentage of damaged acrosomes as revealed by the lectin binding assay. In mammals, these changes are often associated with sperm capacitation. Our observations support the notion that this process may also occur in saurian. While sperm analysis is a valuable method for assessing certain functional changes, additional approaches are required to validate this process.

The activins and inhibins are glycoproteins with a role in the follicular development of vertebrates, that are found in follicular fluid and somatic follicular cells, with a different pattern among taxa. The principal function of activin (Act) is to modulate the follicle-stimulating hormone (FSH) synthesis and secretion, whereas inhibin (Inh) downregulates it. Both factors are modulators of intraovarian follicular recruitment, oocyte maturation, cell proliferation, and steroidogenic activity. Our aim was to characterize the immunolocalization of Act and Inh in the ovarian follicles during the reproductive cycle of the lizard Sceloporus torquatus. Act was detected in the granulosa cells and oocyte cortex in the different stages of follicular development. On the other hand, we identified Inh in the oocyte cortex and the cytoplasm of pyriform and small cells of previtellogenic follicles. Also, we found immunoreactivity in the oocyte cortex, theca, and small cells of vitellogenic and preovulatory follicles. Our data provide evidence that Act and Inh have changes related to the stage of follicular development. This dynamic appears to be conserved among vertebrates and is fundamental to ensure an adequate follicular development in this specie.

While most lizard species are polygamous, dominant males typically have greater access to females and copulate more frequently than submissive ones. Several studies suggest that applying assisted reproduction techniques to this taxon could be an effective tool to enhance conservation programs. Therefore, the proper selection of males for assisted breeding programs is crucial for their overall success. In this study, we report for the first time data on semen and sperm quality from dominant males of the Mexican lizard Sceloporus torquatus using techniques commonly employed in assessing mammalian fertility. The semen and sperm characteristics were as follows: volume, 14.0 µL; sperm concentration, 125.7 × 10⁶ cells/mL; wave motion, 2.7; total motility, 87.8%; sperm viability, 89.0%; normal morphology, 88.8%; plasma membrane integrity, 87.7%; low plasma membrane fluidity, 94.9%; capacitation status: uncapacitated (F), 90.5%; capacitated (B), 7.2%; sperm with acrosome reaction (AR), 2.3%; and, acrosome integrity, 88.8%. These results suggest that, according to mammalian fertility parameters, dominant males of S. torquatus are fertile. This series of tests provides a valuable tool for conducting a comprehensive analysis of the functional changes that may occur in sperm handling during assisted processes, and that may contribute—along with the proper selection of individuals—to reproductive success.