Characterization of sperm and implications for male fertility in the last of the Rhynchocephalians

Abstract

Managing a species of conservation concern can be best achieved when there is information on the reproductive physiology of both sexes available; however, many species lack this critical, baseline information. One such species, the tuatara (Sphenodon punctatus), is the last surviving member of one of the four reptile orders (Rhynchocephalia) and is the only reptile known to lack a male intromittent organ. Culturally and evolutionarily significant, the conservation of this species is a global priority for the maintenance of biodiversity. In light of this, we characterized the morphology, viability and swim speed of mature tuatara sperm for the first time. We found that tuatara sperm are filiform and bear the remarkably conserved three-part sperm structure seen across the animal kingdom. Tuatara sperm are long (mean total length 166 μm), with an approximate head:midpiece:tail ratio of 15:1:17. While tuatara sperm are capable of high levels of within-mating viability (94.53%), the mean viability across all samples was 58.80%. Finally, tuatara sperm had a mean curvilinear velocity swim speed (μ × s − 1) of 82.28. At the population level, there were no differences in viability or mean swim speed between sperm collected from a male’s first mating of a season and repeat matings; however, the maximum sperm swim speed increased in observed repeated matings relative to first matings. Interestingly, faster sperm samples had shorter midpieces, but had greater viability and longer head and tail sections. This work expands our understanding of male reproductive characteristics and their variation to a new order, provides wild references for the assessment of captive individuals, lays the groundwork for potential assisted reproductive techniques and highlights variation in male reproductive potential as an important factor for consideration in future conservation programs for this unique species.

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.…”

“…When a courting pair was seen, we took notes on the duration of any stolzer Gang observed and qualitative descriptions of behaviours made by the courting pair. These data were collected as part of a larger study on reproductive physiology in male tuatara (Lamar et al, 2023); thus, if a mating was found elsewhere in the field, we ceased observation of other courting behaviours and collected biological samples from the mating pair. For this reason, the total durations of some courting behaviours observed are not known.…”

“…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.…”

“…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.…”