Initial collection, characterization, and storage of tuatara (Sphenodon punctatus) sperm offers insight into their unique reproductive system

Abstract

Successful reproduction is critical to the persistence of at-risk species; however, reproductive characteristics are understudied in many wild species. New Zealand’s endemic tuatara (Sphenodon punctatus), the sole surviving member of the reptile order Rhynchocephalia, is restricted to 10% of its historic range. To complement ongoing conservation efforts, we collected and characterized mature sperm from male tuatara for the first time. Semen collected both during mating and from urine after courting contained motile sperm and had the potential for a very high percentage of viable sperm cells (98%). Scanning electron microscopy revealed a filiform sperm cell with distinct divisions: head, midpiece, tail, and reduced end piece. Finally, our initial curvilinear velocity estimates for tuatara sperm are 2–4 times faster than any previously studied reptile. Further work is needed to examine these trends at a larger scale; however, this research provides valuable information regarding reproduction in this basal reptile.

Novel mating behaviours in male tuatara (Sphenodon punctatus) expand our understanding of reptile courtship

“…Finally, of the 69 instances where a male successfully secured a mating opportunity, five appeared to be 'dry'. We define these as matings that were behaviourally typical, with courting being observed beforehand and an adequate amount of time for sperm deposition to have occurred elapsing before interruption (>5 min) (Lamar et al, 2021(Lamar et al, , 2023; however, no sperm or seminal fluid of any kind was present on the outside of or just inside either tuatara's cloacae. In all five of these instances the male shuddered shortly after mounting the female, a presumed indication of sperm ejaculation in this species.…”

“…Finally, we report that 11% of observed matings appeared dry. Three of the five observed dry mating events were observed from the start of mating, when the male mounted the female; in these instances, we can be sure that the pair was not prematurely interrupted (Lamar et al, 2021(Lamar et al, , 2023. Individuals were allowed to mate for between 5 and 10 min after discovery, and in all cases no seminal fluid was found on or in the cloacal opening of the female.…”

“…Once we encountered a mating, we hand captured both individuals and collected any semen present on either's vent, or just inside in the cloacal opening, using a sterile syringe (Lamar et al, 2023). When a mating was observed from the point of the male mounting the female, the pair was allowed to copulate for between 5 and 10 min before being interrupted for sperm collection (Lamar et al, 2021(Lamar et al, , 2023. When mating males did not have any existing permanent identification (crest beads, historic toe codes, or a passive integrated transponder: PIT tag), we injected a PIT tag into the subcutaneous space along the flank.…”

Common goals, different stages: the state of the ARTs for reptile and amphibian conservation

“…We note many sperm cryopreservation protocols developed to date have used sperm collected postmortem, due to the large quantities and quality of sperm obtained and the ease of protocol development this confers (Young et al 2017(Young et al , 2021(Young et al , 2022Campbell et al 2021aCampbell et al , 2021bSandfoss et al 2021). However, encouragingly, two recent studies have looked at sperm collected non-lethally, including the first study on tuatara (Lamar et al 2021;Hobbs et al 2022). We also believe that further studies on fundamental reptile reproductive ecology and physiology, along with testing fundamental hypotheses around cryopreserving reptile reproductive cells and tissues (Campbell et al 2021a) will aid with these conservation and research goals.…”

“…Encouragingly, while the majority of ARTs studies to date have been focussed on squamates and the saltwater crocodile, a recent study reported the first collection, characterisation, and storage of tuatara (Sphenodon punctatus) sperm (Lamar et al 2021). Although preliminary, the authors reported low levels of live sperm recovery post-thaw using the commercial cryoprotectant Synth-a-Freeze™ (containing 10% DMSO buffered with HEPES), making it the first ART study for the ancient reptile order Rhynchocephalia (Lamar et al 2021). That means there has now been at least one published study on ARTs in all four of the extant reptile orders, a small but important step forward in reptile conservation.…”