An artificial uterus is a next-generation incubator used in the medical care of premature birth fetuses and conservation breeding of viviparous animals in public zoos and aquaria [e.g., (1)]. Recent studies by the authors proposed a new artificial uterine system for viviparous elasmobranchs (sharks and batoids). This method is characterized by the usage of “artificial uterine fluid,” a urea-containing liquid that has osmotic pressure and salinity nearly equivalent to those of shark blood plasma (2). In the first application of this method at the Okinawa Churaumi Aquarium (Okinawa, Japan) from 2020 to 2021, two embryonic slendertail lantern sharks, Etmopterus molleri, were successfully maintained for ~5 months until they reached normal birth size (2). These specimens, however, failed to adapt to a seawater environment after “delivery.” Thereafter, in the second round of incubation from 2021 to 2022, an alternative seawater adaptation protocol was applied to the embryos, allowing the stable maintenance of juveniles after delivery (3).

Despite the high efficacy of artificial uterine fluid, as confirmed in previous studies, one of the concerns with this fluid is its applicability to early- to mid-term embryos. Considering the previous studies focused only on late-term embryos—from the very late period of stage 32 to the end of stage 34, the applicability of the fluid to embryos at earlier stages currently remains hypothetical (2). It is generally accepted that embryonic physiological mechanisms dynamically change with its development. Studies on the oviparous cloudy catshark (Scyliorhinus torazame) showed that the mechanisms of osmoregulation, respiration, and nutrient absorption shift from embryonic mode to post-hatched mode in the middle of embryonic development when the respiratory slits of the egg capsule open [e.g., (4–6)]. Similar physiological shifts are also likely to occur in viviparous sharks, and if so, it is possible that the environmental conditions applied to late-term embryos in Tomita et al. (2) cannot be used to maintain earlier-term embryos.

This study reports a third-round incubation experiment on a slendertail lantern shark conducted from 2022 to 2023. This experiment is distinguished from the previous experiments in that it started the incubation half a year earlier in the development process than the previous experiments.

This study is the first to confirm that the artificial uterine fluid developed by Tomita et al. (2) has the ability to maintain mid-term (stage 31) embryos. The 355 days of embryonic incubation achieved in this study is possibly the longest reliable record for viviparous elasmobranchs and other vertebrates (1, 8, 9). Although there are no published data on the gestation period of slendertail lantern sharks, it was assumed that the gestation period of the species was ~1.5 years. This is based on the observation that pregnant females caught in November, 6 months before incubation commenced, include ovulated eggs with no visible embryo (probable stage 1–2 embryo; TT, pers. obs.). Considering this gestational period, the incubation period of the study corresponded to approximately two-thirds of the entire gestational period. This estimate is supported by data from the oviparous sharks Scyrhiorhinus canicula and Chilloscyllium punctatum, whose embryonic development from stages 31–34 accounts for 62 and 68% of the entire developmental process, respectively (7, 10).

Despite the success of long-term embryonic incubation, the technical issue of high mortality in mid-term embryos due to pressure ulcers persists. Such death ceased in embryos after the late portion of stage 32, where the body movement became strong enough to lift and rotate the external yolk sac, suggesting that the removal of permanent pressure on the yolk sac is effective in avoiding this problem. Based on this expectation, the container with the embryo was gently shaken to detach the external yolk sac surface from the container wall once or twice a week after the first case of pressure ulcer was observed. This procedure, however, did not stop the symptoms, and its efficacy could not be quantitatively evaluated in this study. This problem may be avoided in future studies by coating the inner wall of the container with a soft and low-friction material such as mucin hydrogel, which surrounds the embryo in the egg capsule of oviparous elasmobranchs (11).

The present study sheds light on the poorly understood uterine function of the lantern shark, especially the nutrient supply to embryos. This development in elasmobranchs was originally found in myliobatiform stingrays. The inner wall of the uterus is covered with numerous filamentous structures (trophonemata) that secrete lipid-rich “milk”. The embryo grows to a large size at the magnitude of 1,680–4,900% in weight by drinking this fluid [e.g., (12)]. Similar nutrient supply functions were later confirmed in a wide range of elasmobranch taxa, although the amount and composition of nutrients and secretion mechanisms vary greatly among different taxa [e.g., (13)]. The only study on this function in lantern sharks was conducted by Cotton et al. (14). They compared the organic weight of embryonic great lantern sharks (Etmopterus princeps) at different developmental stages and imply the possibility of some level of nutrient transfer from the mother to the embryo. The pattern of organic weight change is, however, determined not only by the amount of maternal nutrient input but also by other factors, such as gestation period and embryonic metabolic rate. Their hypothesis, thus, remains controversial. Interestingly, the incubated embryos that did not acquire any external nutrients grew to ~15 cm in total length. This size matches the natural birth size reported for this species (15). It is, therefore, likely that the maternal nutrient supply, when present, was small in amount in the slender-tail lantern shark, and its absence had little impact on the embryonic growth of this species. In species in which the uterus exhibits strong nourishing capabilities, it will be necessary to add a nutrient supply function to the current artificial uterine system, a task our research group is currently focusing on.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethical review and approval were not required for the animal study because the maintenance and handling of fish in this study were conducted in strict accordance with the guidelines for animal experiments of the Okinawa Churashima Foundation, with the same consideration of animal care and welfare as that for “higher” vertebrates (reptiles, birds, and mammals), as per the guidelines stipulated. The approval from the Institutional Animal Care and Use Committee of Okinawa Churashima Foundation, which is required for higher vertebrates, is waived for “lower” vertebrates including fishes. The studies were conducted in accordance with the local legislation and institutional requirements.

TT: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft. MT: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing – review & editing. AK: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – review & editing. KMu: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review & editing. KMi: Data curation, Formal analysis, Investigation, Validation, Writing – review & editing. KS: Conceptualization, Methodology, Supervision, Validation, Writing – review & editing.