Coral Reefs https://doi.org/10.1007/s00338-022-02268-1 NOTE Conservation of coral reef fishes: a field‑hardy method to cryopreserve spermatogonial cells Jessica Bouwmeester1,2  · Jonathan Daly1,2  · E. Michael Henley1,2  · Lynne R. Parenti3  · Diane E. Pitassy3  · Mary Hagedorn1,2   Received: 24 October 2021 / Accepted: 19 April 2022 © The Author(s) 2022 Abstract The biodiversity of marine fishes is threatened Keywords Asterropteryx semipunctata · Collection globally by climate change and other anthropogenic activi- protocol · Cryopreservation · Primordial germ cells · ties, particularly in coral reef ecosystems. We present a sim- Testes · Vouchers ple, field-hardy method to cryopreserve marine fish gonads, targeting spermatogonial cells (undifferentiated diploid germ cells) with the ultimate goal of permitting recovery of threat- Introduction ened species and populations via gonadal diploid germ cell transplantation technologies. The use of a simplified cryo- Marine and freshwater fishes worldwide provide vital eco- preservation extender based on L-15 medium resulted in system functions such as a source of food, nutrient cycling, minimal decline in spermatogonial cell viability post-thaw. food web dynamics regulation, and increased resilience in Moreover, we compared post-cryopreservation viability of coral reef ecosystems (Hughes 1994; Holmlund and Ham- sperm and spermatogonial cells from gonads cryopreserved mer 1999; Bellwood et al. 2004). Yet the world’s ~ 35,000 with freshly prepared cryoprotectant and with cryoprotect- recognized species of fishes in freshwater and marine eco- ant prepared in advance and stored at −20 °C. We found no systems are under severe threat from habitat fragmentation significant difference, suggesting that these solutions may be and degradation, introduction of exotic species, pollution, prepared in advance and frozen, ready for later use. We urge overfishing, and climate change (Arthington et al. 2016). In conservation, academic, and regulatory agencies to cryobank particular, coral reefs support over a quarter of all marine fish gonads as part of their sample collection processes to life in the oceans, but reef habitats are severely threatened support the biodiversity and security of valuable marine fish (GCRMN 2021). As these reef habitats decline, so does resources, alongside other restoration efforts. coral reef fish diversity and abundance (Jones et al. 2004; Pratchett et al. 2008). We focus on a simple field-hardy method to secure the Topic Editor Alastair Harborne biodiversity and genetic diversity of fishes using modern Supplementary Information The online version contains reproductive technology: spermatogonial cell cryopreserva- supplementary material available at https:// doi. org/ 10. 1007/ tion. These cells are early stage male germ cells that are s00338- 022- 02268-1. still diploid, having not yet undergone meiosis, and retain Jessica Bouwmeester the ability to undergo both oogenesis and spermatogenesis. * bouwmeesterj@si.edu This method is based on the pioneering work of Dr. Yoshi- 1 Smithsonian Conservation Biology Institute, Front Royal, zaki et al. (2010, 2011); Lee et al. (2013, 2016), who revo- VA 22360, USA lutionized fish conservation by defining the parameters of 2 Hawaii Institute of Marine Biology, Kaneohe, HI 96744, fish spermatogonial cell cryopreservation, including thawing USA and implantation into sterile hosts (for details see Yoshi- 3 zaki et al. 2011; ESM 1, Fig. S1). The extraordinary power Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, of this process resides in the ability of the spermatogonial DC 20013-7012, USA cells to readily withstand the cryopreservation process and to Vol.:(012 3456789) Coral Reefs migrate and produce donor gonads in the sterile host. Given purchased worldwide, and freezing these solutions ahead of these spermatogonial cells are able to differentiate into either time, would reduce the efficacy of the cryopreservation pro- testes or ovaries depending on the gender of the host fish cess. Our goal was to simplify the cryopreservation process they are transplanted into, both male and female gametes so that these methods could be incorporated into the work- are recovered, thus facilitating reproduction and the produc- flow of museums, federal agencies and non-governmental tion of donor offspring. Conveniently, the same species is organizations involved in field collections of marine fishes. not needed as the host, so extinct species could potentially be resurrected (Takeuchi et al. 2004; Okutsu et al. 2007; Yoshizaki and Yazawa 2019). Methods Much of the work of Yoshizaki et al. was accomplished in freshwater fishes, which may easily be cultured and Fish collection reared to allow for extensive transplantation studies (Okutsu et al. 2007; Lee et al. 2016; Octavera and Yoshizaki 2020). Twenty-four male Asterropteryx semipunctata (Starry goby) Because the lifecycle of most marine fishes has not been were collected live from a sandy reef flat on the north east closed in captivity (Chen et al. 2020), cryopreserved and of Moku o Loʻe (Coconut Island, GPS: 21.435070° N, thawed marine fish spermatogonial cells cannot yet be tested 157.786901° W) in southern Kāneʻohe Bay, Oʻahu, Hawaiʻi, for their efficacy to produce donor offspring. Therefore, post- over several days in November 2019, and October–Decem- thaw survival of the cells, determined by live/dead staining ber 2020 (see ESM 2 for further details). Fish were identi- and flow cytometry, is one of the few alternative metrics fied and sexed visually after being captured in small hand to test the cryopreservation success of spermatogonial cells nets. Females were released, and males were transported from marine fish species (Figueroa et al. 2016). The broad within 5 min to the laboratory at the Hawaiʻi Institute of survival and use of these thawed spermatogonial cells in Marine Biology (HIMB). Maintenance and handling of live freshwater species support the notion that frozen and thawed fish met the animal care standards of the National Institute marine spermatogonial cells could be successfully trans- of Health (NIH). Full details of the study approval are listed planted (Okutsu et al. 2006; Yoshizaki et al. 2011; Yoshizaki with the Smithsonian Institution, USNM IACUC (approval and Lee 2018), thus producing an effective conservation tool ID #2018-03) and the University of Hawaiʻi IACUC (pro- once the supportive marine husbandry methods have been tocol ID# 12-1491). Fish were collected under permit SAP- devised (de Siqueira-Silva et al. 2018; Yoshizaki and Lee 2020-25 and SAP-2021-33 from the Department of Land 2018). and Natural Resources, Hawaiʻi. Cryopreservation is a low cost, practical method to help conserve aquatic species diversity (Tiersch and Green 2011; Specimen preparation Magnotti et al. 2018). We focus on a simple, rapid and straightforward method to cryopreserve marine fish sper- Live fish were immersed in a 0.01% solution of buffered matogonial cells. The method can potentially help restore tricaine methanesulfonate (MS-222; Sigma-Aldrich, fish populations from the samples stored for many years Inc., MO) dissolved in seawater until gill movement and (Lee et al. 2013), thereby reducing extinction pressures on response to mechanical stimulation ceased (~ 5  min). fish populations. Recently, coral sperm cryopreserved for Individuals were rinsed with seawater and placed dor- over 10 yrs was used to facilitate assisted gene flow between sal surface down on a damp sponge or paper towel. Size populations of a threatened species, Acropora palmata (see was recorded for each individual (Standard Length: the Hagedorn et al. 2021), suggesting that these frozen assets, straight-line distance from the tip of the snout to the base securely stored in biorepositories, will be valuable both on of the caudal fin; ESM 2). The gonad of male gobioid short and long time scales for restoration. Long-term success fishes comprises the testis and accessory gonadal struc- will obviously depend on whether the impacts to fishes and tures, following Miller (1984, 1992). The testis has a sper- their habitats worldwide are being addressed and managed matogenic function, whereas the accessory gonadal struc- appropriately, but this method provides the option to revive tures have a secretory function (Cole and Parenti, 2022). fish populations and species at any time in the future. We surgically removed the gonads from the adult male Here, we present our modification of the methods of specimens, following a standard protocol (Hagedorn et al., Yoshizaki et al. (2010, 2011); Lee et al. (2013, 2016) and 2018). Briefly, an incision was made with micro-dissecting Hagedorn et al. (2018) to accommodate coral reef fishes. scissors to expose the abdominal cavity. Once the gonads Previously, the buffers used to cryopreserve spermatogo- were visible, they were gently peeled off, leaving behind nial cells were complex and had to be freshly made in the the main testicular blood vessel and the mesorchium. The laboratory thereby limiting remote field use. We consid- entire dissection was performed under a stereomicro- ered whether using standard solutions that could be easily scope (Wild M5). Each gonad was weighed and placed 1 3 Coral Reefs into a labelled 1.8 ml cryovial with 500 µl of L15 medium Cryopreservation, bio‑banking, and thawing (Sigma-Aldrich L1518-500ml) and held at 0 °C, on ice. Each pair of testes and accessory gonadal structures was Gonads were treated as defined in Fig. 1. The intact gonads then used in one of two experiments that analyzed the effi- in 500 µl L15 medium destined for cryopreservation received cacy of the field protocol: (1) determine the difference in 500 µl of double strength cryoprotectant (CPA) and were left viability between fresh and cryopreserved testicular cells on ice for an hour of equilibration during which some water (both sperm and spermatogonial cells) using freshly made left the gonad cells and some CPA penetrated the cells. The cryoprotectants, or (2) determine whether there was a dif- CPA solution was prepared at double strength for a final ference in cell viability when cryopreserved tissues were concentration of 0.1 M trehalose, 9.2% v/v DMSO (1.3 M), exposed to freshly made or previously frozen cryoprotect- 10% v/v Fetal Bovine Serum (FBS), in L15 medium after the ants. If successful, this second experiment would ensure initial 1:1 dilution with the sample. The CPA was prepared that solutions could be made ahead of time, frozen and fresh or prepared in advance and frozen at −20 °C until use. taken into the field to help standardize and expedite the (The preparation used is mentioned for each result.) Cryo- cryopreservation process. vials were then placed in a passive cooling device with a Fig. 1 Flow diagram for the fish gonad cryopreservation and preparation for flow cytometer analysis 1 3 Coral Reefs cooling rate of 1 °C per minute (BioCision Cool Cell, Bath, the flow cytometry filters 533/30 (FL1) and 670 LP (FL3), the UK) and moved to a −80 °C freezer until they reached respectively. −80 °C (about 80 min). The samples were then quenched in liquid nitrogen and transferred to a liquid nitrogen Biobank Statistical analyses at −196 °C for at least 24 h. Samples to be thawed were retrieved from the Biobank Gonads from eight fish specimens were used to determine and transferred to a small cryogenic Dewar containing liquid the difference in viability between fresh and cryopreserved nitrogen. They were thawed and assessed one at a time. Each testicular cells (with one gonad used for each treatment), vial was transferred into a water bath at 30 °C and gently and gonads from another eight fish specimens were used to stirred until completely thawed (about 2 min). The intact determine whether there was a difference in cell viability gonad was then transferred to a new tube containing 1 ml when cryopreserved tissues were exposed to freshly made L15 medium, which was renewed every 20 min until an hour or previously frozen cryoprotectants. All data are expressed had passed. The gonad was then ready for dissociation and as cell concentration means (cells per ml) ± standard error. assessment. Due to variation in standard length and gonad size among males, cell concentrations were normalized against gonad Cell dissociation and assessment of sperm weight prior to statistical analyses. All statistical analyses and spermatogonial cell viability were conducted in R (R Core Team 2019) with the pack- ages ggplot2, rcompanion, Rmisc, scales. Cell concentra- Gonads contain sperm cells and spermatogonial cells that tions were log-transformed to fulfil normality assumptions. are the largest present (~ 10 µm) and that can be sorted Cell concentrations from fresh and cryopreserved gonads from other cells by their size (forward scatter) and granu- were compared using a paired t-test, with 95% confidence larity (size scatter), visualized with a flow cytometer (Kise interval (α = 0.05). et al. 2012; Ichida et al. 2017; Hagedorn et al. 2018; Rivers et al. 2020). Sperm and spermatogonial cells were either assessed fresh or post-thaw after cryopreservation. The Results and discussion fresh or cryopreserved and thawed gonads were dissociated in L15 medium with a 0.6-ml glass homogenizer until the Simplified cryopreservation protocol solution appeared homogenous without any visible clumps. The dissociated cells were then filtered through a 40-µm The success profile for these experiments is defined in Fig. 2. cell-straining basket, diluted in L15 medium for a total The total number of all dissociated testicular cells (defined of 990 µl, stained with 5 µl SYBR-14 and 5 µl propidium iodide (PI) fluorescent stains (live/dead sperm viability kit, Invitrogen, Carlsbad, CA), and incubated in the dark, at room temperature. The stained dissociated cells were then analyzed with flow cytometry (BD Accuri C6 Plus Flow Cytometer equipped with a 488 nm excitation laser; Fig. 1) at a maximum rate of 104 cell counts per second. The cell concentration for flow cytometry analysis was maintained below 5 × 106 cells/ml, with samples diluted and re-analyzed if needed. A size vs. granularity profile of spermatogonial and sperm cells was plotted in the BD Accuri C6 Plus soft- ware in a forward scatter vs. side scatter plot and measured against standard-sized beads (2, 5 and 10 µm in diameter) run through the flow cytometer. The forward scatter profile of the 10-µm beads and the lower values of the side scatter profile was used to set a gate corresponding approximately with type A spermatogonial cells (Schulz et al. 2010; Kise et al. 2012; Rivers et al. 2020), which are the target cells for transplantation, and separating them from sperm cells, which made up > 90% of the cells present from analyses. Fig. 2 Fresh versus cryopreserved gonads: difference in concen- Within each category (spermatogonial or sperm cells), live tration of total testicular cells (sperm cells + spermatogonial cells, including intact, damaged, and dead cells), intact sperm cells, and cells were distinguished from dead cells according to their intact spermatogonial cells. The y-axis is plotted on a log scale. Error fluorescent staining with SYBR-14 and PI, detected with bars represent standard error of the mean 1 3 Coral Reefs as all sperm cells and spermatogonial cells, including intact, damaged, and dead cells) was similar between the fresh and cryopreserved gonads (paired t-test, t(15) = 1.13, p = 0.28; Fig. 2; ESM 3, Table S1), indicating no overall loss of cells from the cryopreservation process. When analyzing sperm and spermatogonial cells separately, the number of intact sperm cells was slightly reduced after cryopreservation (paired t-test, t(15) = 2.41, p = 0.03), but the number of intact spermatogonial cells was unaffected by the cryopreserva- tion (paired t-test, t(15) = 0.77, p = 0.46; Fig. 2). On average, spermatogonial cells from fresh gonads comprised only a small fraction of the total testicular cells (0.39%; ESM 3, Table S1), but cryopreservation seemed to have little impact on these overall numbers. These results suggest that the simplification of the solution using standard buffers, such as L15 medium, had little impact on the resulting number of successfully cryopreserved and thawed spermatogonial cells. Given its simplicity, this method may be readily imple- mented in the field by biologists after a brief period of train- ing. This will allow for greater standardization of the cryo- Fig. 3 Cryopreservation using freshly prepared versus frozen cryo- preservation process. The gonads may be cryopreserved as protectant (CPA): difference in the concentration of total testicular cells (sperm cells + spermatogonial cells, including intact, damaged, whole and the testicular cells dissociated later, after thawing. and dead cells), intact sperm cells, and intact spermatogonial cells. For later transplantation purposes, the spermatogonial cells The y-axis is plotted on a log scale. Error bars represent standard would then need to be isolated from the other cells to maxi- error of the mean mize transplantation success, for example, using cell sorting with flow cytometry light scattering (Kise et al. 2012; Ichida reef ecosystems are at even higher risk, due to the increasing et al. 2017; Rivers et al. 2020). pressure of climate change on the reef-building corals that sup- port these ecosystems (Wilson et al. 2008). The challenge is to Cryoprotectant solution prepared in advance get people mobilized to begin this conservation process now, and frozen to incorporate it into collection workflow processes (Hagedorn et al. 2018), and to create additional space and support for The use of frozen cryoprotectant solutions prepared in national cryorepositories. If proper metrics, voucher samples, advance yielded no difference in the concentration of total high-resolution images, and DNA samples are included in sperm and spermatogonial cells (paired t-test, t(7) = −0.02, this process, natural history museums may be willing to hold p = 0.99), intact sperm cells (paired t-test, t(7) = −0.11, these frozen assets for their country of origin. It is important to p = 0.92), or intact spermatogonial cells (paired t-test, cryopreserve and bank these reproductive spermatogonial cells t(7) = −0.52, p = 0.62), (Fig. 3, ESM 3, Table S2) compared now, while they are available, even though current larviculture to freshly prepared cryoprotectant solutions. Therefore, for and aquaculture techniques in most marine fishes are not yet field campaigns on boats or in remote locations, cryopro- sufficiently advanced to proceed with restoring these popula- tectants can be prepared in advance and frozen until needed tions following the methods by Yoshizaki et al. (2011). Once in the field. Additionally, this means the solutions can be culture techniques allow for the full life cycle of marine fishes prepared safely in the laboratory, by laboratory-trained to occur in a controlled captive environment, frozen material personnel. will be available to verify that spermatogonial cell transplanta- tion works equally well for marine fishes as it does for freshwa- Implications for marine fishes conservation ter fishes, which will allow for active fishes restoration efforts to be initiated using already the cryopreserved spermatogonial Regardless of the aquaculture and larviculture husbandry chal- cells. With rapidly advancing research, this may be forthcom- lenges still facing marine species (Vadstein et al. 2018; Chen ing. For example, groupers are ecologically and commercially et al. 2020; Groover et al. 2021), conservation of fishes must be valuable fishes in coral reef ecosystems, and many are listed as considered a high priority due to the severe extinction pressure threatened due overfishing and threats to their habitat (Olsen on many species and populations. This is a crisis for biodi- and LaPlace 1979; Sadovy de Mitcheson et al. 2020). Some versity as well as worldwide food resources (Srinivasan et al. of the key challenges in grouper aquaculture include its life 2010; McCauley et al. 2015; Yan et al. 2021). Fishes in coral history parameters such as longevity and late sexual maturity. 1 3 Coral Reefs Yet, the brown-marbled grouper Epinephelus fuscoguttatus, permitted by statutory regulation or exceeds the permitted use, you will a relatively well-studied and widely cultured grouper, lives need to obtain permission directly from the copyright holder. To view a well in aquaculture and can reach sexual maturity within three copy of this licence, visit http://c reati vecom mons.o rg/l icens es/b y/4.0 /. years in captivity (Sugama et al. 2012; Mustafa et al. 2015; Boonanuntanasarn et al. 2016; Villanueva et al. 2021). This makes it a suitable surrogate candidate for transplantation References by cryopreserved spermatogonia from other grouper species Arthington AH, Dulvy NK, Gladstone W, Winfield IJ (2016) Fish with higher longevity and later sexual maturity. Techniques conservation in freshwater and marine realms: status, threats and for transplantation of cryopreserved spermatogonial cells into management. Aquat Conserv 26:838–857 a surrogate host involve transplantation into the host during Bellwood DR, Hughes TP, Folke C, Nystrom M (2004) Confronting its larval stage, when the endogenous primordial germ cells the coral reef crisis. Nature 429:827–833Boonanuntanasarn S, Bunlipatanon P, Ichida K, Yoohat K, Mengyu are migrating and before they are completely surrounded by O, Detsathit S, Yazawa R, Yoshizaki G (2016) Characterization gonadal somatic cells (Takeuchi et al. 2009; Yazawa et al. of a vasa homolog in the brown-marbled grouper (Epinephelus 2010; Morita et al., 2015). This timing has recently been fuscoguttatus) and its expression in gonad and germ cells dur- established in the brown-marbled grouper using vasa gene ing larval development. Fish Physiol Biochem 42:1621–1636Chen JY, Zeng C, Jerry DR, Cobcroft JM (2020) Recent advances of expression during larval development, with an identified marine ornamental fish larviculture: broodstock reproduction, transplantation window between 9 and 21 days post-hatching live prey and feeding regimes, and comparison between demer- (Boonanuntanasarn et al. 2016). The next step will be to inves- sal and pelagic spawners. Rev Aquac 12:1518–1541 tigate techniques to micro-inject transplanted spermatogonial Cole KS, Parenti LR (2022) Gonad morphology of Rhyacichthys aspro (Valenciennes, 1837), and the diagnostic reproductive cells into the grouper larvae, which are known to be generally morphology of gobioid fishes. J Morph 283:255–272 fragile and difficult to raise (Boonanuntanasarn et al. 2016). de Siqueira-Silva DH, Saito T, dos Santos-Silva AP, da Silva CR, Without critical actions taken to secure the biodiversity of Psenicka M, Yasui GS (2018) Biotechnology applied to fish fishes, fish populations and some of the more vulnerable fish reproduction: tools for conservation. Fish Physiol Biochem 44:1469–1485 species will continue to dwindle and head towards extinction. Figueroa E, Valdebenito I, Farias JG (2016) Technologies used in We urge conservation, academic, and regulatory agencies the study of sperm function in cryopreserved fish spermatozoa. to take up this challenge and secure fish genetic and species Aquacult Res 47:1691–1705 diversity, alongside the other measures being undertaken to GCRMN (2021) The sixth status of coral reefs of the world: 2020 report. In: Souter D, Planes S, Wicquart J, Logan M, Obura D, reduce the anthropogenic pressures on these organisms, both Staub F (eds) on a global and regional scale. Groover EM, Alo MM, Ramee SW, Lipscomb TN, Degidio J-ML, DiMaggio MA (2021) Development of early larviculture pro- Acknowledgements This work was supported by the Paul M. Angell tocols for the melanurus wrasse Halichoeres melanurus. Aqua- Family Foundation, the William H. Donner Family Foundation, the culture 530:735682 Barrett Family Foundation, the Skippy Frank Foundation, the Comp- Hagedorn MM, Daly JP, Carter VL, Cole KS, Jaafar Z, Lager CV, ton Foundation, the Cedar Hill Foundation, the Mastriani Family, the Parenti LR (2018) Cryopreservation of fish spermatogonial DeWitt Family and the Anela Kolohe Foundation. Additional support cells: the future of natural history collections. Sci Rep 8:1–11 was provided by the Smithsonian Conservation Biology Institute, the Hagedorn M, Page CA, O’Neil KL, Flores DM, Tichy L, Conn National Museum of Natural History, the Smithsonian Women’s Com- T, Chamberland VF, Lager C, Zuchowicz N, Lohr K (2021) mittee and the Hawaiʻi Institute of Marine Biology. The fish in Fig- Assisted gene flow using cryopreserved sperm in critically ure 1 was photographed by Dr. Zeehan Jaafar, National University of endangered coral. Proc Natl Acad Sci 118:e2110559118 Singapore. The authors also thank Dr. Yoshizaki for discussion about Holmlund CM, Hammer M (1999) Ecosystem services generated by this process and Mariko Quinn and Riley Perry for their assistance in fish populations. Ecol Econ 29:253–268 catching gobies. This manuscript is Hawaiʻi Institute of Marine Biol- Hughes TP (1994) Catastrophes, phase shifts, and large-scale degra- ogy contribution # (xxx). dation of a Caribbean coral reef. Science 265:1547 Ichida K, Kise K, Morita T, Yazawa R, Takeuchi Y, Yoshizaki G Declarations (2017) Flow-cytometric enrichment of Pacific bluefin tuna type A spermatogonia based on light-scattering properties. Theriog- enology 101:91–98 Conflict of interest On behalf of all the authors, the corresponding Jones GP, McCormick MI, Srinivasan M, Eagle JV (2004) Coral author declares that there is no conflict of interest. decline threatens fish biodiversity in marine reserves. Proc Natl Acad Sci 101:8251–8253 Open Access This article is licensed under a Creative Commons Kise K, Yoshikawa H, Sato M, Tashiro M, Yazawa R, Nagasaka Y, Attribution 4.0 International License, which permits use, sharing, adap- Takeuchi Y, Yoshizaki G (2012) Flow-cytometric isolation and tation, distribution and reproduction in any medium or format, as long enrichment of teleost type A spermatogonia based on light- as you give appropriate credit to the original author(s) and the source, scattering properties. Biol Reprod 86(107):101–112 provide a link to the Creative Commons licence, and indicate if changes Lee S, Iwasaki Y, Shikina S, Yoshizaki G (2013) Generation of func- were made. The images or other third party material in this article are tional eggs and sperm from cryopreserved whole testes. Proc included in the article’s Creative Commons licence, unless indicated Natl Acad Sci 110:1640–1645 otherwise in a credit line to the material. If material is not included in Lee S, Katayama N, Yoshizaki G (2016) Generation of juvenile the article’s Creative Commons licence and your intended use is not rainbow trout derived from cryopreserved whole ovaries by 1 3 Coral Reefs intraperitoneal transplantation of ovarian germ cells. Biochem Sugama K, Rimmer M, Ismi S, Koesharyani I, Suwirya K, Giri N, Biophys Res Commun 478:1478–1483 Alava V (2012) Hatchery management of tiger grouper (Epinephe- Magnotti C, Cerqueira V, Lee-Estevez M, Farias JG, Valdebenito lus fuscoguttatus): a best-practice manual. Australian Centre for I, Figueroa E (2018) Cryopreservation and vitrification of fish International Agricultural Research (ACIAR), Canberra, Australia semen: a review with special emphasis on marine species. Rev Takeuchi Y, Yoshizaki G, Takeuchi T (2004) Surrogate broodstock Aquac 10:15–25 produces salmonids. Nature 430:629–630 McCauley DJ, Pinsky ML, Palumbi SR, Estes JA, Joyce FH, Warner Takeuchi Y, Higuchi K, Yatabe T, Miwa M, Yoshizaki G (2009) RR (2015) Marine defaunation: animal loss in the global ocean. Development of spermatogonial cell transplantation in Nibe Science 347:1255641 croaker, Nibea mitsukurii (Perciformes, Sciaenidae). Biol Reprod Miller P (1984) The tokology of gobioid fishes. In: Potts G, Wooton 81:1055–1063 R (eds) Fish reproduction: strategies and tactics. Academic Tiersch TR, Green CC (eds) (2011) Cryopreservation in aquatic spe- Press, London, pp 119–153 cies, 2nd edition. World Aquaculture Society, Advances in World Miller PJ (1992) The sperm duct gland: a visceral synapomorphy for Aquaculture, Baton Rouge, Louisiana, 1003 pp gobioid fishes. Copeia 1992:253–256 Vadstein O, Attramadal KJ, Bakke I, Forberg T, Olsen Y, Verdegem M, Morita T, Morishima K, Miwa M, Kumakura N, Kudo S, Ichida K, Giatsis C, Skjermo J, Aasen IM, Gatesoupe F-J (2018) Managing Mitsuboshi T, Takeuchi Y, Yoshizaki G (2015) Functional sperm the microbial community of marine fish larvae: a holistic perspec- of the yellowtail (Seriola quinqueradiata) were produced in the tive for larviculture. Front Microbiol 9:1820 small-bodied surrogate, jack mackerel (Trachurus japonicus). Mar Villanueva EG, Hoevenaars K, van Beijnen J, Gonzales AP, Dolorosa Biotechnol 17:644–654 RG, Creencia LA (2021) Protocol Development for the Improved Mustafa S, Hajini MH, Senoo S, Kian AYS (2015) Conditioning of Hatchery Propagation of Tiger Grouper Epinephelus fuscoguttatus broodstock of tiger grouper, Epinephelus fuscoguttatus, in a recir- (Forsskål, 1775) in Palawan, Philippines. The Palawan Scientist culating aquaculture system. Aquaculture 2:117–119 13:132–147 Octavera A, Yoshizaki G (2020) Production of Chinese rosy bitterling Wilson SK, Fisher R, Pratchett MS, Graham N, Dulvy N, Turner R, offspring derived from frozen and vitrified whole testis by sper- Cakacaka A, Polunin NV, Rushton S (2008) Exploitation and matogonial transplantation. Fish Physiol Biochem 46:1431–1442 habitat degradation as agents of change within coral reef fish Okutsu T, Yano A, Nagasawa K, Shikina S, Kobayashi T, Takeuchi Y, communities. Glob Change Biol 14:2796–2809 Yoshizaki G (2006) Manipulation of fish germ cell: visualization, Yan HF, Kyne PM, Jabado RW, Leeney RH, Davidson LN, Derrick DH, cryopreservation and transplantation. J Reprod Dev 52:685–693 Finucci B, Freckleton RP, Fordham SV, Dulvy NK (2021) Over- Okutsu T, Shikina S, Kanno M, Takeuchi Y, Yoshizaki G (2007) Pro- fishing and habitat loss drive range contraction of iconic marine duction of trout offspring from triploid salmon parents. Science fishes to near extinction. Sci Adv 7:eabb6026 317:1517–1517 Yazawa R, Takeuchi Y, Higuchi K, Yatabe T, Kabeya N, Yoshizaki Olsen DA, LaPlace J (1979) A study of a Virgin Islands grouper fish- G (2010) Chub mackerel gonads support colonization, survival, ery based on a breeding aggregation. Proc Gulf Carib Fish Inst and proliferation of intraperitoneally transplanted xenogenic germ 31:130–144 cells. Biol Reprod 82:896–904 Pratchett MS, Munday PL, Wilson SK, Graham NA, Cinner JE, Bell- Yoshizaki G, Lee S (2018) Production of live fish derived from fro- wood DR, Jones GP, Polunin NV, McClanahan TR (2008) Effects zen germ cells via germ cell transplantation. Stem Cell Res of climate-induced coral bleaching on coral-reef fishes: ecologi- 29:103–110 cal and economic consequences. Oceanogr Mar Biol 46:251–296 Yoshizaki G, Yazawa R (2019) Application of surrogate broodstock R Core Team (2019) R: A Language and Environment for Statisti- technology in aquaculture. Fish Sci 85:429–437 cal Computing. R Foundation for Statistical Computing, Vienna, Yoshizaki G, Ichikawa M, Hayashi M, Iwasaki Y, Miwa M, Shikina Austria. https:// www.R- proje ct. org S, Okutsu T (2010) Sexual plasticity of ovarian germ cells in Rivers N, Daly J, Jones R, Temple-Smith P (2020) Cryopreservation of rainbow trout. Development 137:1227–1230 testicular tissue from Murray River Rainbowfish, Melanotaenia Yoshizaki G, Fujinuma K, Iwasaki Y, Okutsu T, Shikina S, Yazawa R, fluviatilis. Sci Rep 10:1–9 Takeuchi Y (2011) Spermatogonial transplantation in fish: a novel Sadovy de Mitcheson YJ, Linardich C, Barreiros JP, Ralph GM, Agu- method for the preservation of genetic resources. Comp Biochem ilar-Perera A, Afonso P, Erisman BE, Pollard DA, Fennessy ST, Physiol Part D Genomics Proteomics 6:55–61 Bertoncini AA (2020) Valuable but vulnerable: Over-fishing and under-management continue to threaten groupers so what now? Publisher’s Note Springer Nature remains neutral with regard to Mar Policy 116:103909 jurisdictional claims in published maps and institutional affiliations. Schulz RW, de França LR, Lareyre J-J, LeGac F, Chiarini-Garcia H, Nobrega RH, Miura T (2010) Spermatogenesis in fish. Gen Comp Endocrinol 165:390–411 Srinivasan UT, Cheung WW, Watson R, Sumaila UR (2010) Food security implications of global marine catch losses due to over- fishing. J Bioeconomics 12:183–200 1 3