{"paper_id":"23221f48-be55-4f93-9d4d-cbb03910cfa8","body_text":"Celiotomy to collect spermatogonial stem cells in Brycon orbignyanus for species preservation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Celiotomy to collect spermatogonial stem cells in Brycon orbignyanus for species preservation Laís Gonçalves Silva, Karel Gelina Torres-Lozano, Rômulo Batista Rodrígues, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6681336/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Surgical techniques in aquatic species are underdeveloped, despite these species comprising a significant superclass among vertebrates. In the context of species preservation, studies involving the use of reproductive tissues typically involve the euthanasia of donors. Thus, in animals at high risk of extinction, the practice of sacrificing them presents a point of contradiction between ex situ conservation efforts. The objective of this study was to compare the viability of spermatogonia stem cells (SSCs) collected from Brycon orbignyanus using conventional methods (euthanasia) versus a surgical procedure. Lateral celiotomy was performed on 27 immatures males to obtain a portion of gonadal tissue. The fish were divided into three groups (n = 9), with each group receiving polyglactin 910, polyester, or catgut sutures, respectively, for celiorrhaphy. Dermorraphy was performed using nylon sutures in all groups. A. An additional euthanasia group consisted of nine animals exposed to 20 mg/L of propofol. The survival rate over a 48-hour period was 100% for the polyglactin 910 group and 77.7% for the polyester and catgut groups. Additionally, the viability of SSCs was similar between the euthanasia and surgical procedures. The lateral celiotomy technique is feasible for obtaining SSCs in fish with laterally compressed anatomy, such as B. orbignyanus . Additionally, the technique allows the preservation of SSCs as a model for endangered fish species. Endangered species gonadectomy SSC surgery piracanjuba Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The agenda for strategies regarding the conservation and sustainable use of aquatic genetic resources (AqGR) is considered fundamental by the Food and Agriculture Organization (FAO 2022 ). Among these conservation strategies, in situ preservation of wild and farmed fish for reproduction and genetic material collection is of utmost importance. Similarly, ex situ conservation emphasizes the need to establish germplasm banks, not only for species bred in captivity but also for wild species (FAO 2019 ). The use of gametes in the establishment of fish germplasm banks can be crucial for preserving the DNA of animals with high zootechnical value (Thelie et al. 2019 ) or species at risk of extinction (Hagedorn et al. 2018 ; Swaminathan et al. 2020 ). Genetic variability within a population and the adaptive genetic traits of a species depends on both the male and female genome. In this context, spermatogonial steam cells (SSCs) emerge as an alternative for preserving genetic diversity, given their potential for transplantation into other fish. Due to their genetic plasticity, SSCs have the ability to differentiate into both oocytes and sperm, depending on the somatic environment in which they develop (Lee et al., 2013 ; Okutsu et al. 2006 ). Among the potential uses of SSCs, animal reproduction, production of transgenic animals and conservation of endangered species are recurrently highlighted applications (Oatley 2018 ; Honaramooz and Yang 2010 ). In fish, the use of SSCs is almost exclusively related to the use in the transplantation of undifferentiated cells to another recipient fish, which is called a \"surrogate mother\" (Wylie et al. 2023 ). The use of SSCs from genetic preservation, allows the supply of reproductive material throughout year-round, facilitating juvenile fish production (Abualreesh et al. 2020 ) in addition to transplantation into adult fish (Majhi et al. 2020 ; Xie et al. 2020 ). To access SSCs, it is necessary to obtain a fragment of gonadal tissue, which is routinely collected after euthanasia (Hagedorn et al., 2018 ; Rivers et al., 2020 ; Rosa et al., 2023 ). Even when ethical standards are strictly followed, sacrificing the animal poses a challenge to in situ preservation strategies. The growing interest in using fish as experimental models, combined with the development and performance of surgical procedures, has been timidly explored. Surgical techniques commonly used in terrestrial animals can be adapted for fish (Sladky and Clarke III, 2016 ). The first report of gonadectomy via lateral celiotomy dates to the 1960s (Jhons and Liley 1970 ). However, there has been a notable absence of reports on surgical techniques to fish with laterally compressed anatomy that does not allow access via the ventral region, such as Brycon orbignyanus . Numerous species of the genus Brycon are listed and classified at different levels of threat regarding their wild populations. And this scenario is no different for B. orbignyanus , which is classified as a critically endangered species (ICMBio, 2018 ). The almost complete reduction of wild stocks of the species has been reduced in almost its entirety (Oliveira et al., 2017 ; Agostinho et al., 2008 ). And studies have related the need for strategies to protect the species, either in situ (Tonella et al., 2019 ) or ex situ (Galo et al., 2019 ). As for the ex-situ strategy, the recent study by Siqueira-Silva et al. ( 2021 ) showed viability from the transplantation of SSCs. The objective of this study was to compare the viability of spermatogonia stem cells (SSCs) collected from B. orbignyanus using conventional methods (euthanasia) versus a surgical procedure. Furthermore, it demonstrates the successful development of the protocol in an endangered species (Siqueira-Silva et al. 2021 ). Material and methods Ethics statement This study was conducted with the established guidelines of the Conselho Nacional de Controle e Experimentação Animal – CONCEA (National Council for Control and Animal Experimentation) and by the approval of the Animal Use Ethics Committee of the Federal University of Rio Grande do Sul (CEUA-UFRGS), project number 44839. Fish maintenance and experimental conditions Thirty-six males B. orbignyanus , were randomly selected from CEMIG aquaculture station, located in Itutinga, (Minas Gerais - Brazil). The animals were five years old, with an average weight and length of 452.51 ± 101.20 g and 33.91 ± 2.48 cm, respectively, and stocked in a breeding maintenance tank at a density of one animal per 3 m2. After selection, the animals were kept in the laboratory in three 1000 L aquariums at 22°C, with constant water renewal for the experiment. E xperimental design To access testicular tissue and collect spermatogonial stem cells (SSCs) in fish with laterally compressed anatomy, such as piracanjuba, the surgical method described by Jhons and Liley (1970) was used. After collecting SSCs, three types of threads were tested for suturing the B. orbignyanus (Figure 1). Thus: C1 - polyglactin 910 3-0, C2-polyester 3-0 and C3- catgut 3-0 sutures for celiorrhaphy after unilateral partial gonadectomy. Nine animals were used in each experimental group. The SSCs that were obtained by surgery in experiment 1 (Ec) were compared with the SSCs obtained by the conventional method, euthanasia (Ee) (Figure 2). Anesthesia The fish were collected from the aquariums and transferred to an induction tank with 12 L of aquarium water mixed with propofol (Propovan, Cristália-São Paulo) at a concentration of 2.5mg/L (Oda et al. 2014). Behaviors such as longitudinal axis rotation, loss of the escape reflex to fin pinching, and reduced opercular movements were monitored to ensure the correct anesthetic depth for surgery (Schottger and Julin 1967). After induction, the fish’s length (cm) and weight (g) were measured. The fish were then transferred to a 4 L aquarium containing 1.5 mg/L propofol for anesthetic maintenance. The animals were then positioned in right lateral decubitus position on a surgical bed made of foam covered with cotton fabric, with a silicone hose attached to an aquarium pump delivering 220 L/h of water into the oral cavity for anesthetic maintenance. Dissolved oxygen (5.5 mg/L) was supplied, and water circulated through the opercula. The animal’s eyes were covered with wet tissue to protect the ocular surface, and the body was draped with a sterile, non-woven surgical drape (Polarfix -São Paulo) to maintain moisture during the procedure. Before surgery, 0.5 mg/kg of morphine, 7 mg/kg of enrofloxacin, and 2.2 mg/kg of flunixin were administered intramuscularly (Martin et al. 2021) in the lateral region near the dorsal fin. Surgical Procedure The area was prepared using a surgical drape, and scales were removed in a 1 cm by 1 cm area along the lateral midline, from the posterior part of the pectoral fin to one-third of the total pelvic fin length. The area was disin- fected with 0.92% saline solution. An incision was made with a #15 scalpel blade, extending approximately 5 cm in a cranio-caudal direction, 0.5 cm below the lateral midline (Figure 3A). Muscular tissue was separated using Met- zenbaum scissors, and the ribs (3 rd and 4 th ) were sectioned with blunt-tipped scissors to access the coelomic cavity (Figure 3B). The left gonad, located in the mid-cranial portion (Figure 3C), was partially excised (0.5 cm fragment) for SSC extraction (Figure 3D). Celiorrhaphy was performed using a simple continuous suture to approximate the muscle with the materials specific to each treatment group (C1, C2, or C3), followed by dermorrhaphy using a Sultan suture pattern with non-absorbable 3-0 nylon in all groups. Surgical recovery After surgery for C1, C2 and C3 animals, each fish was placed in a recovery tank containing 20 L of water to monitor the complete recovery from anesthesia, confirmed by coordinated swimming16. After recovery, the fish were transferred to a tank with 1% saline solution for five minutes. The animals were then transferred to experimental tanks to monitor the healing of surgical wounds for 48 hours, as well as to observe possible mortalities. After this time, all animals were returned to the breeding tank to reduce the stress of confinement and minimize mortality. Over the course of another 120 hours, the presence of dead animals was observed in this breeding tank. Euthanasia The fish in group Ec were euthanized by immersion in a propofol solution at 20 mg/L, followed by spinal cord section after opercular movements ceased (Balko et al. 2018). Validation analyzes for SSCs The SSC collection followed the protocols of Abualreesh et al. (2020) and Franek et al. (2019), with modifications. Gonadal fragments from each group were placed in 15 mL falcon tubes with 10 mL Leibovitz L-15 medium (Sigma-Aldrich-USA) pH 7.8 to maintain hydration. They were exposed to a 0.5% hypochlorite solution for two minutes, washed three times with L-15, dried, weighed, and sectioned into 3 X 3 mm fragments. Three fragments were placed in 2.0 mL microtubes and cut into smaller pieces with scissors. For enzymatic digestion, 50 μL of 0.25 mg/mL trypsin (Dinâmica Química-Brazil) + 0.02 mg/mL EDTA (Neon comercial-São Paulo) was added to the microtubes and incubated on a shaking platform for 1.30 h. To stop enzyme activity, 400 μL of L-15 and 100 μL of fetal bovine serum (FBS) were added. The solution was filtered through a 40 μm nylon filter into 1.5 mL microtubes and centrifuged at 500G for 20 minutes. The supernatant was discarded, and the cell pellet was resuspended in 300 μL of L-15 + 10% FBS. Membrane integrity of the spermatogonial cells was assessed according to Franek et al. (2019). A mixture of 10 μL of the cell suspension and 10 μL of 0.4% trypan blue was placed in a Neubauer hemocytometer. Five fields of the hemocytometer were analyzed under a microscope (Nikon E200, Tokyo, Japan) at 40X magnification, and live cells were counted in triplicate. Statistical analysis Data were analyzed using normality tests (Shapiro-Wilk and Kolmogorov-Smirnov) and homogeneity tests (Bartlett). Parametric data were analyzed by one-way analysis of variance (One-Way ANOVA), and when sig- nificant differences were observed, means were compared using Tukey’s test. Parametric data are presented as bar graphs (mean ± standard deviation). Non-parametric data were analyzed using the Kruskal-Walli’s test, and differences among groups were tested by Dunn’s test. Non-parametric data are presented in Box-Whisker plots (mean, minimum, and maximum values). Analyses and graph creation were performed using GraphPad Prism 9.0 software. Results Experiment 1: There was no significant difference regarding the weight and length of the animals that made up the experimental groups, as well as for the surgery time using the different types of threads tested for suturing the animals (Figure 4A-C). In a descriptive analysis, there was no difference between the groups in the immediate postoperative period concerning the extent of the surgical wound for strategic access to the gonad. All groups showed ease in locating the target tissue, minimal muscle wall bleeding during the procedure, and satisfactory edge coaptation (Figure 5). After 36 hours of postoperative monitoring, gelatinous structures with a cotton-like appearance when immersed in water and a yellowish coloration were observed on the fins, eyes, oral cavity, and surgical site. Despite the presence of these structures over the surgical incision site, none of the animals showed suture dehiscence or wound opening during the observation period (Figure 6). Survival was recorded up to 48 hours after surgery across the groups (Figure 7A). During the first 36 hours, no deaths were recorded in any group. At the 48-hour evaluation, groups C2 and C3 showed a mortality rate of 22.2% (n=2) each (Figure 7B). Experiment 2: When comparing viability among spermatogonia obtained by the surgical method and the euthanasia method, no statistical difference was observed between the methods, as shown in Figure 8. Discussion The efficacy of lateral celiotomy for unilateral partial gonadectomy presented in this study, brings a novel alternative for collection of spermatogonial steam cell, aiming the conservation of an endangered fish species. However, surgical procedures in species with compressed anatomy is challenged, due the difficult to have access into the celomatic cavity. The wide diversity of particularities among species is possibly the main challenge for those managing these animals, whether for aquariums, production, or conservation purposes 29 . The initial challenge imposed in the development of the surgical technique that emerged for spermatogonia collection was the laterally compressed anatomy of B. orbignyanus . Studies describing the use of surgery in fish are scarce and, with the exception of Jhons and Liley ( 1970 ), the surgical access incision is always recommended in the ventral region of the animal. This can be observed in the studies of Brown & Richards ( 1979 ) 30 (Salmo salar and Salmo trutta ), Chapman and Park ( 2005 ) ( Acipenser oxyrinchus de sotoi ) and Majhi et al. ( 2020 ) ( Clarias magur ). Even in books (McLean 1994 ; Johnson 2000 ) or reviews (Wildgoose 2000 ) that address surgery in fish, the suggested access is always ventral. It is worth noting that the species related in these studies have a fusiform anatomy, that is, a flattening of the ventral region, facilitating surgical access. The laterally compressed anatomy of B. orbignyanus presents significant difficulty in accessing the celomatic cavity due to the ventral junction of the animal flattened sides. The lateral access method described by Jhons and Liley ( 1970 ) for Trichogaster trichopterus was used to facilitate the localization of SCC’s, which are in the most dorsal portion of the coelomic cavity in these animals, ensuring safety in the application of the gonadectomy technique. This is consistent with the premise of surgical procedures in terrestrial animals, as noted by Sladky and Clarke III ( 2016 ), which must be adapted to the species and utilize materials commonly used in planned surgeries. The time required for the gonadectomy procedure via lateral access applied to B. orbignyanus was another relevant point in the development of the methodology proposed in this study. It is important to understand that this is an aquatic animal and necessarily requires an adapted structure to survive outside its comfortable environment. The surgical procedure took between 11–12 minutes, regardless of the suture used, which is comparable to the time reported by Majhi et al. ( 2020 ) for C. Magnum using ventral access. Additionally, in S. salar and O. mykiss , the average time was 15 minutes, also using ventral access (McLean 1994 ). After the surgical procedure, there was considerable consistency in the approximation of the incision sites across the different sutures tested. The polyglactin 910 3 − 0 suture used in the C1 group was also employed by Chapman and Park (2011) and was associated with good surgical outcome. On the other hand, Majhi et al. ( 2020 ) reported great efficiency using catgut 3 − 0 sutures for sperm collect in Clarias magnum , achieving complete healing within four weeks. None of the animals exhibited external suture openings with the nylon 3 − 0 suture during the 48 hours observation period, indicating an effective healing process across all groups. This finding is comparable to the study by Bockelmann et al. (2010), which involved amputating the caudal fin of Cyprinus carpio . In that study, surgical wound healing occurred with the formation of an apical epidermal layer within the first two postoperative days. In fish, stress is influenced by environmental and physical factors, including management, dominance, crowding, aggression, and water quality. These factors lead to immune suppression, which directly affects fish survival (Tort, 2011 ). B. orbignyanus is a sensitive species, and like many others, they are negatively affected by confinement and show improvement once released (Johnson 2000 ). During the 48-hour postoperative observation period, the C1 group showed 100% survival rate, while the C2 and C3 groups showed a 77.7% survival rate, respectively. Once relocated to breeding tank no additional deaths were reported. Germplasm banks are a strategic tool for the conservation of ex situ genetic resources. Cryopreservation of SSCs has the potential to save species anywhere, as spermatogonia plasticity is considered a valuable tool in combating species extinction (Hagerdon et al. 2018). Commonly, protocols for collecting spermatogonial cells in different species for cryopreservation are developed with the euthanasia of the animals, as described for Asterropteryx semipunctata (Hagerdon et al. 2018), Melanotaenia fluviatilis (Rivers et al. 2020 ) and Rhamdia quelen (Rosa et al. 2023 ). In our study, we observed no difference in the number of viable SSCs collected from animals undergoing surgery compared to those collected through euthanasia, indicating that surgical procedures can be a viable alternative for obtaining these cells without sacrificing the animals. In this study, for the first time an efficient method of lateral celiotomy to collect SSCs in a fish species with a laterally compressed anatomy. This technique can be replicated in other species with the same anatomical structure, thereby preserving the life of the fish, which often needs to be sacrificed for SSC collection. It is important to emphasize that B. orbignyanus is a species classified as critically endangered 18 and urgently requires effective conservation strategies. In this sense, greater care must be taken with each of these specimens, even though their role as SSC donors is preponderant to, for example, recover threatened populations from gene banks. In conclusion, lateral celiotomy was efficient in collecting SSCs from a species with laterally compressed anatomy, avoiding the sacrifice of B. orbignyanus, classified as endangered. Declarations Acknowledgments We would like to thank Research and Development Program regulated by ANEEL, BAESA – Energética Barra Grande for their financial support [grant number P&D 03936-1410/2021 - Desenvolvimento de biotecnologia para recuperação populacional de espécie nativa de peixe em reservátorio de UHE - Case: Piracanjuba (Brycon orbignyanus )]. We would like to express our gratitude to CEMIG - Itutinga Hydroelectric Station (MG) for pro- viding the animals and the facilities for conducting this study. We also acknowledge the support of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [grant number 23038.011255/2021–17 and 88887.877624/2023-00]. Additionally, we extend our gratitude to research fellows Danilo P. Streit Jr. (CNPq grant 305387/2022-7), Romulo Batista Rodrigues (CNPq grants 141717/2019-0 and 200285/ 2021-1), Thaiza Rodrigues de Freitas (CNPq grant 141423/2021-8) and Jayme Aparecido Povh (CNPq Grant 312072/2021-0) from the National Council for Scientific and Technological Development (CNPq-Brazil). We also warmly thank the members of the AQUAM Laboratory at UFRGS. Author Contributions Laís Gonçalves da Silva: conducted the surgical management, identified suitable medical records, data collection, initial drafting; Karel Gelina Torres-Lozano: conducted the data collection, executed practical activities for validation tests, drafting and revised the final text, graphical design, interpretation data; Rômulo Batista Rodrígues: design of the study, statistical analyze, elaborated graphs, interpretation data, draft review; Thaiza Rodrigues de Freitas: data collection, conception and design, executed practical activities for validation tests, interpretation data; Jayme Aparecido Povh: acquisition and interpretation data, critical review; Louise Nex Spica: data collection, draft review; Nathalia dos Santos Teixeira: data collection, draft review; Douglas Cosme Selle: data collection, provided intraoperative photographs; Jhony Lisbôa Benato: provided intraoperative photographs, graphical designs; Raquel Santos dos Santos: draft review; Eduardo Thomé Nicoleti: draft review; Renata Villar Dantas: draft review; Danilo Pedro Streit Jr.: orientation and monitoring all the experimental activities, provided the final writing and critical review to approved the manuscript, and all authors provided a critical review of the manuscript and endorse the final version. Founding The authors would express their grateful to the Research and Development Program regulated by ANEEL, BAESA – Energética Barra Grande for their financial support [grant number P&D 03936-1410/2021 - Desenvolvimento de biotecnologia para recuperaç ã o populacional de espécie nativa de peixe em reservatório de UHE - case: piracanjuba (Brycon orbignyanus) ] . We would thank to the support of the FAPERGS (projeto: 21/2551-0001369-4). Also thank to the Companhia Energética de Minas Gerais (CEMIG) for the conception of the fishes and use of the laboratory. Finally, a special thanks to the members of AQUAM research group. Competing interests The authors declare that there are no competing interests regarding report. 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Mol Ecol Resour 25:e13868. https://doi.org/10.1111/1755-0998.13868 Xie X, Nóbrega R, Pšenička M (2020) Spermatogonial stem cells in fish: characterization, isolation, enrichment, and recent advances of in vitro culture systems. Biomolecules 10:644. https://doi.org/10.3390/biom1004 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6681336\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":460383680,\"identity\":\"d5882e05-0bbe-491f-8e5f-9ed073999262\",\"order_by\":0,\"name\":\"Laís Gonçalves Silva\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Laís\",\"middleName\":\"Gonçalves\",\"lastName\":\"Silva\",\"suffix\":\"\"},{\"id\":460383683,\"identity\":\"1195dfb6-da6a-4479-bbb8-aae4c4642aea\",\"order_by\":1,\"name\":\"Karel Gelina Torres-Lozano\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Karel\",\"middleName\":\"Gelina\",\"lastName\":\"Torres-Lozano\",\"suffix\":\"\"},{\"id\":460383684,\"identity\":\"2ea8c0ec-3ca4-43b0-a5ab-8573577d4aa5\",\"order_by\":2,\"name\":\"Rômulo Batista Rodrígues\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidade Federal de Santa Maria\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Rômulo\",\"middleName\":\"Batista\",\"lastName\":\"Rodrígues\",\"suffix\":\"\"},{\"id\":460383685,\"identity\":\"cfa14bb2-b820-4d5c-888d-76cc42e1285d\",\"order_by\":3,\"name\":\"Thaiza Rodrigues Freitas\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Thaiza\",\"middleName\":\"Rodrigues\",\"lastName\":\"Freitas\",\"suffix\":\"\"},{\"id\":460383687,\"identity\":\"004871c2-a098-4c4d-911c-4fa7080ad10d\",\"order_by\":4,\"name\":\"Jayme Aparecido Povh\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Mato Grosso do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jayme\",\"middleName\":\"Aparecido\",\"lastName\":\"Povh\",\"suffix\":\"\"},{\"id\":460383688,\"identity\":\"f6dfbf10-085a-4d7a-a277-6c400f587b6e\",\"order_by\":5,\"name\":\"Louise Nex Spica\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Mato Grosso do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Louise\",\"middleName\":\"Nex\",\"lastName\":\"Spica\",\"suffix\":\"\"},{\"id\":460383689,\"identity\":\"2f254657-3262-41cb-ada5-59f719f9a114\",\"order_by\":6,\"name\":\"Nathalia dos Santos Teixeira\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Nathalia\",\"middleName\":\"dos Santos\",\"lastName\":\"Teixeira\",\"suffix\":\"\"},{\"id\":460383690,\"identity\":\"00e0f4a3-e591-469a-b36b-f7ff8798964d\",\"order_by\":7,\"name\":\"Douglas Cosme Selle\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Douglas\",\"middleName\":\"Cosme\",\"lastName\":\"Selle\",\"suffix\":\"\"},{\"id\":460383691,\"identity\":\"3bb4d08f-61e2-4e9c-9e3e-36732c0ee666\",\"order_by\":8,\"name\":\"Jhony Lisbôa Benato\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jhony\",\"middleName\":\"Lisbôa\",\"lastName\":\"Benato\",\"suffix\":\"\"},{\"id\":460383692,\"identity\":\"98464584-b917-4e4a-aa74-c8147be555d5\",\"order_by\":9,\"name\":\"Raquel Santos Santos\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Raquel\",\"middleName\":\"Santos\",\"lastName\":\"Santos\",\"suffix\":\"\"},{\"id\":460383693,\"identity\":\"ed7871a9-d918-4d96-afff-f1dd4f809ae2\",\"order_by\":10,\"name\":\"Eduardo Thomé Nicoleti\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Eduardo\",\"middleName\":\"Thomé\",\"lastName\":\"Nicoleti\",\"suffix\":\"\"},{\"id\":460383694,\"identity\":\"327f843f-3c02-4549-aaa6-d317291d34fc\",\"order_by\":11,\"name\":\"Renata Villar Dantas\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Renata\",\"middleName\":\"Villar\",\"lastName\":\"Dantas\",\"suffix\":\"\"},{\"id\":460383695,\"identity\":\"da28242b-3385-4e5a-a6b9-5a55968c9bc8\",\"order_by\":12,\"name\":\"Danilo Pedro Streit Jr.\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3PvWrDMBDA8SuCdDns9Uz7EIIOHRrwq8hLsoTQbB5K8ZTJ0DVDyJo3yJThhCBeVLJmdAl0yuC8QeV+kQ5R0q0U/RHolh8nAYRCfzF2p/6ec4jbQZwkyt3YzmwhKc4h8EVAj88gUfWsWeWQpldGm/3M0O3TvBaj5XGS2KFiZSEro55ivTB0vdlJMXk9TiQPJGdjUIgoW/JIZEEge8h6905SxLhhPTVEVNV+svnYclEiAuvCkbiQXpK4l7u/UFbajmS76rstKM3EQ6L14KZp8m56WYptkz/cuS3Vy/beQz6jw1nBafCz+LcgFAqF/ntvwJRZ/R6YXd4AAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Federal University of Rio Grande do Sul\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Danilo\",\"middleName\":\"Pedro\",\"lastName\":\"Streit\",\"suffix\":\"Jr.\"}],\"badges\":[],\"createdAt\":\"2025-05-16 13:53:17\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6681336/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6681336/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":83420463,\"identity\":\"8d93a015-ab8c-42b9-b35e-e32f21b46cad\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:50:04\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1286932,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIncision area and treatment groups with the number of \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e and different suture used.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/035a524039f0e8bf8b75aa50.png\"},{\"id\":83420984,\"identity\":\"38ff1fe0-c2b1-4aa3-8d36-66881f74c60d\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:58:06\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1073879,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTreatments using surgical procedure with the Euthanasia group (Ec) to collect testicular tissue and the test validation teste for enzymatic digestion by tripan blue staining to analyzed spermatogonial steam cells (SSC) of \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/020c872a258934b61244e642.png\"},{\"id\":83420476,\"identity\":\"451dcd21-dd43-42ec-9957-e64ec42074ae\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:50:05\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":325036,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Scalpel blade incision. (B) Ribs section with blunt-tipped scissors. (C) Opening of the coelomic cavity. (D) Excision of the fragment of the gonad of \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e to obtain spermatogonial cells.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/a39ee495d1218ad4d4f1db85.png\"},{\"id\":83420468,\"identity\":\"23389fac-e110-47d4-9952-2ffb5d758fd8\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:50:05\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":218276,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCharacteristics of \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e and surgery time. A) Weight (g) (p=0.1690); B) Total length (cm) (p=0.5407); C) Surgery time (min) (p=0.8492). No differences were observed between the experimental groups according to the Kruskal-Walli’s test.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/7d12b2ca832cf214c4771253.png\"},{\"id\":83420497,\"identity\":\"911ae78f-2527-4261-beec-7056e6df2a53\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:50:06\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2344047,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImmediate postoperative period of a \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e specimen.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/a3d630e5970c3c40f41c25d0.png\"},{\"id\":83420479,\"identity\":\"326e79e5-d097-4427-9b40-cf3e4953a596\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:50:05\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":997479,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePresence of a yellowish gelatinous structure over the surgical wound, observed 36 hours postoperatively in \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/93fe80c7abdd9ba29937a97c.png\"},{\"id\":83420472,\"identity\":\"2c6677c4-ab2c-4e76-b905-f03fe9935232\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:50:05\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":217068,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Survival demonstration 48 hours after surgery in \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e. (B) Mortality rate of the groups after the surgical procedure for SSC collection during the observation period.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/02a1619ac9e6044a9a9e7a64.png\"},{\"id\":83420465,\"identity\":\"4dc82a8a-bdd9-425b-8d82-d5d57876ae16\",\"added_by\":\"auto\",\"created_at\":\"2025-05-26 01:50:05\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":99837,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eComparison among the number of cells corrected for 0.05 grams of tissue of \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e, presented in bars. The surgery group is represented in light blue, and the euthanasia group (control) in dark blue.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/79609d72e78517cef8391a99.png\"},{\"id\":84445759,\"identity\":\"24113ffb-adf6-474d-ae19-d45963940a87\",\"added_by\":\"auto\",\"created_at\":\"2025-06-12 05:33:04\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":9345998,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6681336/v1/e207f6f6-6a0c-4d49-bf66-6e64c64ef9db.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Celiotomy to collect spermatogonial stem cells in Brycon orbignyanus for species preservation\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003e\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003eThe agenda for strategies regarding the conservation and sustainable use of aquatic genetic resources (AqGR) is considered fundamental by the Food and Agriculture Organization (FAO \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Among these conservation strategies, in situ preservation of wild and farmed fish for reproduction and genetic material collection is of utmost importance. Similarly, ex situ conservation emphasizes the need to establish germplasm banks, not only for species bred in captivity but also for wild species (FAO \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eThe use of gametes in the establishment of fish germplasm banks can be crucial for preserving the DNA of animals with high zootechnical value (Thelie et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e) or species at risk of extinction (Hagedorn et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Swaminathan et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Genetic variability within a population and the adaptive genetic traits of a species depends on both the male and female genome. In this context, spermatogonial steam cells (SSCs) emerge as an alternative for preserving genetic diversity, given their potential for transplantation into other fish. Due to their genetic plasticity, SSCs have the ability to differentiate into both oocytes and sperm, depending on the somatic environment in which they develop (Lee et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Okutsu et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eAmong the potential uses of SSCs, animal reproduction, production of transgenic animals and conservation of endangered species are recurrently highlighted applications (Oatley \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Honaramooz and Yang \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). In fish, the use of SSCs is almost exclusively related to the use in the transplantation of undifferentiated cells to another recipient fish, which is called a \\\"surrogate mother\\\" (Wylie et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). The use of SSCs from genetic preservation, allows the supply of reproductive material throughout year-round, facilitating juvenile fish production (Abualreesh et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) in addition to transplantation into adult fish (Majhi et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Xie et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTo access SSCs, it is necessary to obtain a fragment of gonadal tissue, which is routinely collected after euthanasia (Hagedorn et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Rivers et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Rosa et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Even when ethical standards are strictly followed, sacrificing the animal poses a challenge to in situ preservation strategies. The growing interest in using fish as experimental models, combined with the development and performance of surgical procedures, has been timidly explored. Surgical techniques commonly used in terrestrial animals can be adapted for fish (Sladky and Clarke III, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). The first report of gonadectomy via lateral celiotomy dates to the 1960s (Jhons and Liley \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e1970\\u003c/span\\u003e). However, there has been a notable absence of reports on surgical techniques to fish with laterally compressed anatomy that does not allow access via the ventral region, such as \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e.\\u003c/p\\u003e\\u003cp\\u003eNumerous species of the genus \\u003cem\\u003eBrycon\\u003c/em\\u003e are listed and classified at different levels of threat regarding their wild populations. And this scenario is no different for \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e, which is classified as a critically endangered species (ICMBio, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). The almost complete reduction of wild stocks of the species has been reduced in almost its entirety (Oliveira et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Agostinho et al., \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). And studies have related the need for strategies to protect the species, either in situ (Tonella et al., \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e) or ex situ (Galo et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). As for the ex-situ strategy, the recent study by Siqueira-Silva et al. (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) showed viability from the transplantation of SSCs.\\u003c/p\\u003e\\u003cp\\u003eThe objective of this study was to compare the viability of spermatogonia stem cells (SSCs) collected from \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e using conventional methods (euthanasia) versus a surgical procedure. Furthermore, it demonstrates the successful development of the protocol in an endangered species (Siqueira-Silva et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e).\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\"},{\"header\":\"Material and methods\",\"content\":\"\\u003ch2\\u003eEthics statement\\u003c/h2\\u003e\\n\\u003cp\\u003eThis\\u0026nbsp;study\\u0026nbsp;was\\u0026nbsp;conducted\\u0026nbsp;with\\u0026nbsp;the\\u0026nbsp;established\\u0026nbsp;guidelines\\u0026nbsp;of\\u0026nbsp;the\\u0026nbsp;Conselho\\u0026nbsp;Nacional\\u0026nbsp;de\\u0026nbsp;Controle\\u0026nbsp;e\\u0026nbsp;Experimenta\\u0026ccedil;\\u0026atilde;o Animal\\u0026nbsp;\\u0026ndash;\\u0026nbsp;CONCEA\\u0026nbsp;(National\\u0026nbsp;Council\\u0026nbsp;for\\u0026nbsp;Control\\u0026nbsp;and\\u0026nbsp;Animal\\u0026nbsp;Experimentation)\\u0026nbsp;and\\u0026nbsp;by\\u0026nbsp;the\\u0026nbsp;approval\\u0026nbsp;of\\u0026nbsp;the\\u0026nbsp;Animal Use\\u0026nbsp;Ethics\\u0026nbsp;Committee\\u0026nbsp;of\\u0026nbsp;the\\u0026nbsp;Federal\\u0026nbsp;University\\u0026nbsp;of\\u0026nbsp;Rio\\u0026nbsp;Grande\\u0026nbsp;do\\u0026nbsp;Sul\\u0026nbsp;(CEUA-UFRGS),\\u0026nbsp;project\\u0026nbsp;number\\u0026nbsp;44839.\\u003c/p\\u003e\\n\\u003ch2\\u003eFish maintenance and experimental conditions\\u003c/h2\\u003e\\n\\u003cp\\u003eThirty-six males \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e, were randomly selected from CEMIG aquaculture station, located in Itutinga,\\u0026nbsp;(Minas Gerais - Brazil). The animals were five years old, with an average weight and length of 452.51\\u003cem\\u003e\\u0026plusmn;\\u003c/em\\u003e101.20 g\\u0026nbsp;and 33.91\\u003cem\\u003e\\u0026plusmn;\\u003c/em\\u003e2.48 cm, respectively, and stocked in a breeding maintenance tank at a density of one animal per 3 m2.\\u003c/p\\u003e\\n\\u003cp\\u003eAfter selection, the animals were kept in the laboratory in three 1000 L aquariums at 22\\u0026deg;C, with constant water renewal for the experiment.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eE\\u003c/strong\\u003e\\u003cstrong\\u003experimental design\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo access testicular tissue and collect spermatogonial stem cells (SSCs) in fish with laterally compressed anatomy, such as piracanjuba, the surgical method described by Jhons and Liley (1970) was used. After collecting SSCs, three types of threads were tested for suturing the \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e (Figure 1). Thus: C1 - polyglactin 910 3-0, C2-polyester 3-0 and C3- catgut 3-0 sutures for celiorrhaphy after unilateral partial gonadectomy. Nine animals were used in each experimental group.\\u003c/p\\u003e\\n\\u003cp\\u003eThe SSCs that were obtained by surgery in experiment 1 (Ec) were compared with the SSCs obtained by the conventional method, euthanasia (Ee) (Figure 2).\\u003c/p\\u003e\\n\\u003ch2\\u003eAnesthesia\\u003c/h2\\u003e\\n\\u003cp\\u003eThe fish were collected from the aquariums and transferred to an induction tank with 12 L of aquarium water mixed with propofol (Propovan, Crist\\u0026aacute;lia-S\\u0026atilde;o Paulo) at a concentration of 2.5mg/L (Oda et al. 2014). Behaviors such as longitudinal axis rotation, loss of the escape reflex to fin pinching, and reduced opercular movements were monitored to ensure the correct anesthetic depth for surgery (Schottger and Julin 1967). After induction, the fish\\u0026rsquo;s length (cm) and weight (g) were measured.\\u003c/p\\u003e\\n\\u003cp\\u003eThe fish were then transferred to a 4 L aquarium containing 1.5 mg/L propofol for anesthetic maintenance. The animals were then positioned in right lateral\\u0026nbsp;decubitus position on a surgical bed made of foam covered with cotton fabric, with a silicone hose attached to an aquarium pump delivering 220 L/h of water into the oral cavity for anesthetic maintenance. Dissolved oxygen (5.5 mg/L) was supplied, and water circulated through the opercula. The animal\\u0026rsquo;s eyes were covered with wet tissue to protect the ocular surface, and the body was draped with a sterile, non-woven surgical drape (Polarfix -S\\u0026atilde;o Paulo) to maintain moisture during the procedure. Before surgery, 0.5 mg/kg of morphine, 7 mg/kg of enrofloxacin, and 2.2 mg/kg of flunixin were administered intramuscularly (Martin et al. 2021)\\u0026nbsp;in the lateral region near the dorsal fin.\\u003c/p\\u003e\\n\\u003ch2\\u003eSurgical Procedure\\u003c/h2\\u003e\\n\\u003cp\\u003eThe area was prepared using a surgical drape, and scales were removed in a 1 cm by 1 cm area along the lateral midline, from the posterior part of the pectoral fin to one-third of the total pelvic fin length. The area was disin- fected with 0.92% saline solution. An incision was made with a #15 scalpel blade, extending approximately 5 cm\\u0026nbsp;in a cranio-caudal direction, 0.5 cm below the lateral midline (Figure 3A). Muscular tissue was separated using\\u0026nbsp;Met- zenbaum\\u0026nbsp;scissors,\\u0026nbsp;and\\u0026nbsp;the\\u0026nbsp;ribs\\u0026nbsp;(3\\u003cem\\u003erd\\u0026nbsp;\\u003c/em\\u003eand\\u0026nbsp;4\\u003cem\\u003eth\\u003c/em\\u003e)\\u0026nbsp;were\\u0026nbsp;sectioned\\u0026nbsp;with\\u0026nbsp;blunt-tipped\\u0026nbsp;scissors\\u0026nbsp;to\\u0026nbsp;access\\u0026nbsp;the\\u0026nbsp;coelomic\\u0026nbsp;cavity\\u0026nbsp;(Figure 3B). The left gonad, located in the mid-cranial portion (Figure 3C), was partially excised (0.5 cm fragment) for\\u0026nbsp;SSC extraction (Figure\\u0026nbsp;3D).\\u003c/p\\u003e\\n\\u003cp\\u003eCeliorrhaphy was performed using a simple continuous suture to approximate the muscle with the materials specific to each treatment group (C1, C2, or C3), followed by dermorrhaphy using a Sultan suture pattern with non-absorbable 3-0 nylon in all groups.\\u003c/p\\u003e\\n\\u003ch2\\u003eSurgical recovery\\u003c/h2\\u003e\\n\\u003cp\\u003eAfter\\u0026nbsp;surgery\\u0026nbsp;for\\u0026nbsp;C1,\\u0026nbsp;C2\\u0026nbsp;and\\u0026nbsp;C3\\u0026nbsp;animals,\\u0026nbsp;each\\u0026nbsp;fish\\u0026nbsp;was\\u0026nbsp;placed\\u0026nbsp;in\\u0026nbsp;a\\u0026nbsp;recovery\\u0026nbsp;tank\\u0026nbsp;containing\\u0026nbsp;20\\u0026nbsp;L\\u0026nbsp;of\\u0026nbsp;water\\u0026nbsp;to\\u0026nbsp;monitor the complete recovery from anesthesia, confirmed by coordinated swimming16. After recovery,\\u0026nbsp;the fish were transferred to a tank with 1% saline solution for five minutes. The animals were then transferred to experimental tanks to monitor the healing of surgical wounds for 48 hours, as well as to observe possible mortalities. After this time, all animals were returned to the breeding tank to reduce the stress of confinement and minimize mortality. Over the course of another 120 hours, the presence of dead animals was observed in this breeding\\u0026nbsp;tank.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEuthanasia\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe fish in group Ec were euthanized by immersion in a propofol solution at 20 mg/L, followed by spinal cord section after opercular movements ceased (Balko et al. 2018).\\u003c/p\\u003e\\n\\u003ch2\\u003eValidation analyzes for SSCs\\u003c/h2\\u003e\\n\\u003cp\\u003eThe SSC collection followed the protocols of Abualreesh et al. (2020) and Franek et al. (2019), with modifications. Gonadal fragments from each group were placed in 15 mL falcon tubes with 10 mL Leibovitz L-15 medium (Sigma-Aldrich-USA) pH 7.8 to maintain hydration. They were exposed to a 0.5% hypochlorite solution for two minutes, washed three times with L-15, dried, weighed, and sectioned into 3 X 3 mm fragments. Three fragments were placed in 2.0 mL microtubes and cut into smaller pieces with scissors.\\u003c/p\\u003e\\n\\u003cp\\u003eFor enzymatic digestion, 50 \\u0026mu;L of 0.25 mg/mL trypsin (Din\\u0026acirc;mica Qu\\u0026iacute;mica-Brazil) + 0.02 mg/mL EDTA (Neon comercial-S\\u0026atilde;o Paulo) was added to the microtubes and incubated on a shaking platform for 1.30 h. To stop enzyme activity, 400 \\u0026mu;L of L-15 and 100 \\u0026mu;L of fetal bovine serum (FBS) were added. The solution was filtered through a 40 \\u0026mu;m nylon filter into 1.5 mL microtubes and centrifuged at 500G for 20 minutes. The supernatant was discarded, and the cell pellet was resuspended in 300 \\u0026mu;L of L-15 + 10% FBS.\\u003c/p\\u003e\\n\\u003cp\\u003eMembrane integrity of the spermatogonial cells was assessed according to Franek et al. (2019). A mixture of 10 \\u0026mu;L of the cell suspension and 10 \\u0026mu;L of 0.4% trypan blue was placed in a Neubauer hemocytometer. Five fields of the hemocytometer were analyzed under a microscope (Nikon E200, Tokyo, Japan) at 40X magnification, and live cells were counted in triplicate.\\u003c/p\\u003e\\n\\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e\\n\\u003cp\\u003eData were analyzed using normality tests (Shapiro-Wilk and Kolmogorov-Smirnov) and homogeneity tests (Bartlett). Parametric data were analyzed by one-way analysis of variance (One-Way ANOVA), and when sig- nificant differences were observed, means were compared using Tukey\\u0026rsquo;s test. Parametric data are presented as \\u0026nbsp;bar graphs (mean \\u003cem\\u003e\\u0026plusmn;\\u0026nbsp;\\u003c/em\\u003estandard deviation). Non-parametric data were analyzed using the Kruskal-Walli\\u0026rsquo;s test, and differences among groups were tested by Dunn\\u0026rsquo;s test. Non-parametric data are presented in Box-Whisker plots (mean, minimum, and maximum values). Analyses and graph creation were performed using GraphPad Prism 9.0 software.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003ch2\\u003eExperiment 1:\\u003c/h2\\u003e\\n\\u003cp\\u003eThere was no significant difference regarding the weight and length of the animals that made up the experimental groups, as well as for the surgery time using the different types of threads tested for suturing the animals (Figure 4A-C).\\u003c/p\\u003e\\n\\u003cp\\u003eIn a descriptive analysis, there was no difference between the groups in the immediate postoperative period concerning the extent of the surgical wound for strategic access to the gonad. All groups showed ease in locating the target tissue, minimal muscle wall bleeding during the procedure, and satisfactory edge coaptation (Figure 5).\\u003c/p\\u003e\\n\\u003cp\\u003eAfter 36 hours of postoperative monitoring, gelatinous structures with a cotton-like appearance when immersed in water and a yellowish coloration were observed on the fins, eyes, oral cavity, and surgical site. Despite the presence of these structures over the surgical incision site, none of the animals showed suture dehiscence or wound opening during the observation period (Figure 6).\\u003c/p\\u003e\\n\\u003cp\\u003eSurvival was recorded up to 48 hours after surgery across the groups (Figure 7A). During the first 36 hours, no deaths were recorded in any group. At the 48-hour evaluation, groups C2 and C3 showed a mortality rate of 22.2% (n=2) each (Figure 7B).\\u003c/p\\u003e\\n\\u003ch2\\u003eExperiment 2:\\u003c/h2\\u003e\\n\\u003cp\\u003eWhen comparing viability among spermatogonia obtained by the surgical method and the euthanasia method, no statistical difference was observed between the methods, as shown in Figure 8.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eThe efficacy of lateral celiotomy for unilateral partial gonadectomy presented in this study, brings a novel alternative for collection of spermatogonial steam cell, aiming the conservation of an endangered fish species. However, surgical procedures in species with compressed anatomy is challenged, due the difficult to have access into the celomatic cavity.\\u003c/p\\u003e \\u003cp\\u003eThe wide diversity of particularities among species is possibly the main challenge for those managing these animals, whether for aquariums, production, or conservation purposes\\u003csup\\u003e29\\u003c/sup\\u003e. The initial challenge imposed in the development of the surgical technique that emerged for spermatogonia collection was the laterally compressed anatomy of \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e. Studies describing the use of surgery in fish are scarce and, with the exception of Jhons and Liley (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e1970\\u003c/span\\u003e), the surgical access incision is always recommended in the ventral region of the animal. This can be observed in the studies of Brown \\u0026amp; Richards (\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e1979\\u003c/span\\u003e)\\u003csup\\u003e30\\u003c/sup\\u003e \\u003cem\\u003e(Salmo salar and Salmo trutta\\u003c/em\\u003e), Chapman and Park (\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e) (\\u003cem\\u003eAcipenser oxyrinchus de sotoi\\u003c/em\\u003e) and Majhi et al. (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) (\\u003cem\\u003eClarias magur\\u003c/em\\u003e). Even in books (McLean \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e1994\\u003c/span\\u003e; Johnson \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e) or reviews (Wildgoose \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e) that address surgery in fish, the suggested access is always ventral. It is worth noting that the species related in these studies have a fusiform anatomy, that is, a flattening of the ventral region, facilitating surgical access.\\u003c/p\\u003e \\u003cp\\u003eThe laterally compressed anatomy of \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e presents significant difficulty in accessing the celomatic cavity due to the ventral junction of the animal flattened sides. The lateral access method described by Jhons and Liley (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e1970\\u003c/span\\u003e) for \\u003cem\\u003eTrichogaster trichopterus\\u003c/em\\u003e was used to facilitate the localization of SCC\\u0026rsquo;s, which are in the most dorsal portion of the coelomic cavity in these animals, ensuring safety in the application of the gonadectomy technique. This is consistent with the premise of surgical procedures in terrestrial animals, as noted by Sladky and Clarke III (\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e), which must be adapted to the species and utilize materials commonly used in planned surgeries.\\u003c/p\\u003e \\u003cp\\u003eThe time required for the gonadectomy procedure via lateral access applied to \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e was another relevant point in the development of the methodology proposed in this study. It is important to understand that this is an aquatic animal and necessarily requires an adapted structure to survive outside its comfortable environment. The surgical procedure took between 11\\u0026ndash;12 minutes, regardless of the suture used, which is comparable to the time reported by Majhi et al. (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) for \\u003cem\\u003eC. Magnum\\u003c/em\\u003e using ventral access. Additionally, in \\u003cem\\u003eS. salar\\u003c/em\\u003e and \\u003cem\\u003eO. mykiss\\u003c/em\\u003e, the average time was 15 minutes, also using ventral access (McLean \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e1994\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eAfter the surgical procedure, there was considerable consistency in the approximation of the incision sites across the different sutures tested. The polyglactin 910 3\\u0026thinsp;\\u0026minus;\\u0026thinsp;0 suture used in the C1 group was also employed by Chapman and Park (2011) and was associated with good surgical outcome. On the other hand, Majhi et al. (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) reported great efficiency using catgut 3\\u0026thinsp;\\u0026minus;\\u0026thinsp;0 sutures for sperm collect in \\u003cem\\u003eClarias magnum\\u003c/em\\u003e, achieving complete healing within four weeks.\\u003c/p\\u003e \\u003cp\\u003eNone of the animals exhibited external suture openings with the nylon 3\\u0026thinsp;\\u0026minus;\\u0026thinsp;0 suture during the 48 hours observation period, indicating an effective healing process across all groups. This finding is comparable to the study by Bockelmann et al. (2010), which involved amputating the caudal fin of \\u003cem\\u003eCyprinus carpio\\u003c/em\\u003e. In that study, surgical wound healing occurred with the formation of an apical epidermal layer within the first two postoperative days.\\u003c/p\\u003e \\u003cp\\u003eIn fish, stress is influenced by environmental and physical factors, including management, dominance, crowding, aggression, and water quality. These factors lead to immune suppression, which directly affects fish survival (Tort, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e is a sensitive species, and like many others, they are negatively affected by confinement and show improvement once released (Johnson \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2000\\u003c/span\\u003e). During the 48-hour postoperative observation period, the C1 group showed 100% survival rate, while the C2 and C3 groups showed a 77.7% survival rate, respectively. Once relocated to breeding tank no additional deaths were reported.\\u003c/p\\u003e \\u003cp\\u003eGermplasm banks are a strategic tool for the conservation of \\u003cem\\u003eex situ\\u003c/em\\u003e genetic resources. Cryopreservation of \\u003cem\\u003eSSCs\\u003c/em\\u003e has the potential to save species anywhere, as spermatogonia plasticity is considered a valuable tool in combating species extinction (Hagerdon et al. 2018). Commonly, protocols for collecting spermatogonial cells in different species for cryopreservation are developed with the euthanasia of the animals, as described for \\u003cem\\u003eAsterropteryx semipunctata\\u003c/em\\u003e (Hagerdon et al. 2018), \\u003cem\\u003eMelanotaenia fluviatilis\\u003c/em\\u003e (Rivers et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) and \\u003cem\\u003eRhamdia quelen\\u003c/em\\u003e (Rosa et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). In our study, we observed no difference in the number of viable \\u003cem\\u003eSSCs\\u003c/em\\u003e collected from animals undergoing surgery compared to those collected through euthanasia, indicating that surgical procedures can be a viable alternative for obtaining these cells without sacrificing the animals.\\u003c/p\\u003e \\u003cp\\u003eIn this study, for the first time an efficient method of lateral celiotomy to collect SSCs in a fish species with a laterally compressed anatomy. This technique can be replicated in other species with the same anatomical structure, thereby preserving the life of the fish, which often needs to be sacrificed for SSC collection. It is important to emphasize that \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e is a species classified as critically endangered\\u003csup\\u003e18\\u003c/sup\\u003e and urgently requires effective conservation strategies. In this sense, greater care must be taken with each of these specimens, even though their role as SSC donors is preponderant to, for example, recover threatened populations from gene banks. In conclusion, lateral celiotomy was efficient in collecting SSCs from a species with laterally compressed anatomy, avoiding the sacrifice of B. orbignyanus, classified as endangered.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eAcknowledgments\\u003c/h2\\u003e\\n\\u003cp\\u003eWe would like to thank Research and Development Program regulated by ANEEL, BAESA \\u0026ndash; Energ\\u0026eacute;tica Barra Grande for their financial support [grant number P\\u0026amp;D 03936-1410/2021 - Desenvolvimento de biotecnologia para recupera\\u0026ccedil;\\u0026atilde;o populacional de esp\\u0026eacute;cie nativa de peixe em reserv\\u0026aacute;torio de UHE - Case: Piracanjuba \\u003cem\\u003e(Brycon orbignyanus\\u003c/em\\u003e)]. We\\u0026nbsp;would like to express our gratitude to CEMIG - Itutinga Hydroelectric Station (MG) for pro- viding the animals and the facilities for conducting this study.\\u0026nbsp;We\\u0026nbsp;also acknowledge the support of Coordena\\u0026ccedil;\\u0026atilde;o de Aperfei\\u0026ccedil;oamento de Pessoal de N\\u0026iacute;vel Superior (CAPES) [grant number 23038.011255/2021\\u0026ndash;17 and 88887.877624/2023-00]. Additionally, we extend our gratitude to research fellows Danilo P.\\u0026nbsp;Streit Jr.\\u0026nbsp;(CNPq\\u0026nbsp;grant 305387/2022-7), Romulo Batista Rodrigues (CNPq grants 141717/2019-0 and 200285/ 2021-1), Thaiza\\u0026nbsp;Rodrigues de Freitas (CNPq grant 141423/2021-8) and Jayme Aparecido Povh (CNPq Grant 312072/2021-0)\\u0026nbsp;from the National Council for Scientific and Technological Development (CNPq-Brazil). We\\u0026nbsp;also warmly thank the members of the AQUAM\\u0026nbsp;Laboratory at\\u0026nbsp;UFRGS.\\u003c/p\\u003e\\n\\u003ch2\\u003eAuthor Contributions\\u003c/h2\\u003e\\n\\u003cp\\u003eLa\\u0026iacute;s Gon\\u0026ccedil;alves da Silva: conducted the surgical management, identified suitable medical records, data collection, initial drafting; Karel Gelina Torres-Lozano:\\u0026nbsp;conducted\\u0026nbsp;the\\u0026nbsp;data\\u0026nbsp;collection,\\u0026nbsp;executed\\u0026nbsp;practical\\u0026nbsp;activities\\u0026nbsp;for\\u0026nbsp;validation\\u0026nbsp;tests,\\u0026nbsp;drafting\\u0026nbsp;and\\u0026nbsp;revised\\u0026nbsp;the\\u0026nbsp;final\\u0026nbsp;text,\\u0026nbsp;graphical\\u0026nbsp;design,\\u0026nbsp;interpretation\\u0026nbsp;data;\\u0026nbsp;R\\u0026ocirc;mulo Batista Rodr\\u0026iacute;gues:\\u0026nbsp;design\\u0026nbsp;of\\u0026nbsp;the\\u0026nbsp;study,\\u0026nbsp;statistical\\u0026nbsp;analyze,\\u0026nbsp;elaborated\\u0026nbsp;graphs,\\u0026nbsp;interpretation\\u0026nbsp;data,\\u0026nbsp;draft\\u0026nbsp;review;\\u0026nbsp;Thaiza Rodrigues de Freitas:\\u0026nbsp;data\\u0026nbsp;collection,\\u0026nbsp;conception\\u0026nbsp;and\\u0026nbsp;design,\\u0026nbsp;executed\\u0026nbsp;practical\\u0026nbsp;activities\\u0026nbsp;for\\u0026nbsp;validation\\u0026nbsp;tests, interpretation data; Jayme Aparecido Povh:\\u0026nbsp;acquisition and interpretation data, critical review;\\u0026nbsp;Louise Nex Spica: data collection, draft review;\\u0026nbsp;Nathalia dos Santos Teixeira: data collection, draft review;\\u0026nbsp;Douglas Cosme Selle: data collection, provided intraoperative photographs; Jhony Lisb\\u0026ocirc;a Benato: provided intraoperative photographs, graphical designs; Raquel Santos dos Santos: draft review;\\u0026nbsp;Eduardo Thom\\u0026eacute; Nicoleti: draft review;\\u0026nbsp;Renata Villar Dantas:\\u0026nbsp;draft review;\\u0026nbsp;Danilo Pedro Streit Jr.: orientation and monitoring\\u0026nbsp;all\\u0026nbsp;the\\u0026nbsp;experimental\\u0026nbsp;activities,\\u0026nbsp;provided\\u0026nbsp;the\\u0026nbsp;final\\u0026nbsp;writing\\u0026nbsp;and\\u0026nbsp;critical\\u0026nbsp;review\\u0026nbsp;to\\u0026nbsp;approved\\u0026nbsp;the\\u0026nbsp;manuscript, and all authors provided a critical review of the manuscript and endorse the final\\u0026nbsp;version.\\u003c/p\\u003e\\n\\u003ch2\\u003eFounding\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors would express their grateful to the Research and Development Program regulated by ANEEL, BAESA \\u0026ndash; Energ\\u0026eacute;tica Barra Grande for their financial support [grant number P\\u0026amp;D 03936-1410/2021 - \\u003cem\\u003eDesenvolvimento de\\u003c/em\\u003e \\u003cem\\u003ebiotecnologia para recupera\\u0026ccedil;\\u003c/em\\u003e\\u0026atilde;\\u003cem\\u003eo populacional de esp\\u0026eacute;cie nativa de peixe em reservat\\u0026oacute;rio de UHE - case: piracanjuba (Brycon orbignyanus)\\u003c/em\\u003e]\\u003cem\\u003e.\\u003c/em\\u003e We would thank to the support of the FAPERGS (projeto: 21/2551-0001369-4). Also thank to the Companhia Energ\\u0026eacute;tica de Minas Gerais (CEMIG) for the conception of the fishes and use of the laboratory. Finally, a special thanks to the members of AQUAM research group.\\u003c/p\\u003e\\n\\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors declare that there are no competing interests regarding report.\\u003c/p\\u003e\\n\\u003ch2\\u003eEthics approval\\u003c/h2\\u003e\\n\\u003cp\\u003eThis study was conducted with the established guidelines of the Conselho Nacional de Controle e Experimenta\\u0026ccedil;\\u0026atilde;o Animal \\u0026ndash; CONCEA (National Council for Control and Animal Experimentation) and by the approval of the Animal Use Ethics Committee of the Federal University of Rio Grande do Sul (CEUA-UFRGS), project number 44839.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eAbualreesh M, Myers JN, Gurbatow J, Johnson A, Xing D, Wang J, Li S, Coogan M, Vo K, Husseini NE, Dunham RA, Butts IAE (2020) Development of a spermatogonia cryopreservation protocol for blue catfish, \\u003cem\\u003eIctalurus furcatus\\u003c/em\\u003e. Cryobiology 97:46\\u0026ndash;52. https://doi.org/10.1016/j.cryobiol.2020.10.010\\u003c/li\\u003e\\n \\u003cli\\u003eAgostinho AA, Zaniboni-Filho E, Lima FCT (2008) \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e (Valenciennes 1850). In: Machado ABM, Drummond GM, Paglia AP (eds) Livro vermelho da fauna brasileira amea\\u0026ccedil;ada de extin\\u0026ccedil;\\u0026atilde;o.\\u0026nbsp;MMA, Funda\\u0026ccedil;\\u0026atilde;o Biodiversitas, Bras\\u0026iacute;lia/Belo Horizonte, pp 54\\u0026ndash;56.\\u003c/li\\u003e\\n \\u003cli\\u003eBalko JA, Oda A, Posner LP (2018) Use of tricaine methanesulfonate or propofol for immersion euthanasia of goldfish (\\u003cem\\u003eCarassius auratus\\u003c/em\\u003e). 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FAO Commission on Genetic Resources for Food and Agriculture Assessments, Rome. https://doi.org/10.4060/CA5256EN\\u003c/li\\u003e\\n \\u003cli\\u003eFAO (2022) Global Plan of Action for the Conservation, Sustainable Use and Development of Aquatic Genetic Resources for Food and Agriculture. Commission on Genetic Resources for Food and Agriculture, Rome. https://doi.org/10.4060/cb9905en\\u003c/li\\u003e\\n \\u003cli\\u003eFraněk R, Marinović Z, Lujić J, Urb\\u0026aacute;nyi B, Fuč\\u0026iacute;kov\\u0026aacute; M, Ka\\u0026scaron;par V, P\\u0026scaron;enička M, Horv\\u0026aacute;th A (2019) Cryopreservation and transplantation of common carp spermatogonia. PLoS One 14:e0205481. https://doi.org/10.1371/journal.pone.0205481\\u003c/li\\u003e\\n \\u003cli\\u003eGalo JM, Streit-Junior DP, Oliveira CA, Povh JP, Fornari DC, Digmayer M, Ribeiro RP (2019) Quality of fresh and cryopreserved semen and their influence on the rates of fertilization, hatching and quality of the larvae of \\u003cem\\u003ePiaractus mesopotamicus\\u003c/em\\u003e. Braz J Biol 79:438\\u0026ndash;445. https://doi.org/10.1590/1519-6984.182391\\u003c/li\\u003e\\n \\u003cli\\u003eHagedorn MM, Daly JP, Carter VL, Cole KS, Jaafar Z, Lager CVA (2018) Cryopreservation of fish spermatogonial cells: The future of natural history collections. Sci Rep 8:6149. https://doi.org/10.1038/s41598-018-24269-3\\u003c/li\\u003e\\n \\u003cli\\u003eHonaramooz A, Yang Y (2010) Recent advances in application of male germ cell transplantation in farm animals. Vet Med Int 2011:657860. https://doi.org/10.4061/2011/657860\\u003c/li\\u003e\\n \\u003cli\\u003eICMBio (2018) Livro Vermelho da Fauna Brasileira Amea\\u0026ccedil;ada de Extin\\u0026ccedil;\\u0026atilde;o.\\u0026nbsp;Instituto Chico Mendes de Conserva\\u0026ccedil;\\u0026atilde;o da Biodiversidade, Bras\\u0026iacute;lia.\\u003c/li\\u003e\\n \\u003cli\\u003eJhons LS, Liley NR (1970) The effects of gonadectomy and testosterone treatment on the reproductive behavior of the male blue gourami \\u003cem\\u003eTrichogaster trichopterus\\u003c/em\\u003e. Can J Zool 48:977\\u0026ndash;987. https://doi.org/10.1139/z70-173\\u003c/li\\u003e\\n \\u003cli\\u003eJohnson G (2000) Surgical techniques. In: Ostrander GK (ed) The laboratory fish. Academic Press, San Diego, pp 557\\u0026ndash;567. https://doi.org/10.1016/B978-012529650-2/50042-1\\u003c/li\\u003e\\n \\u003cli\\u003eLee S, Iwasaki Y, Shikina S, Yoshizaki G (2013) Generation of functional eggs and sperm from cryopreserved whole testes. Proc Natl Acad Sci USA 110:1640\\u0026ndash;1645. https://doi.org/10.1073/pnas.1218468110\\u003c/li\\u003e\\n \\u003cli\\u003eMajhi SK, Maurya PK, Kumar S, Mohindra V, Lal KK (2020) Non-invasive method for collection of \\u003cem\\u003eClarias magur\\u003c/em\\u003e spermatozoa for breeding. MethodsX 7:100929. https://doi.org/10.1016/j.mex.2020.100929\\u003c/li\\u003e\\n \\u003cli\\u003eMartin M, Smith S, Kleinhenz M, Magnin G, Lin Z, Kuhn D, Montgomery S, Coetzee J (2021) Comparative pharmacokinetics and tissue concentrations of flunixin meglumine and meloxicam in tilapia (\\u003cem\\u003eOreochromis spp\\u003c/em\\u003e.). Fishes 6:68. https://doi.org/10.3390/fishes6040068\\u003c/li\\u003e\\n \\u003cli\\u003eMcLean E (1994) Microsurgery: Gonadectomy. In: Hochachka PW, Mommsen TP (eds) Biochemistry and molecular biology of fishes, vol 3. Elsevier, pp 63\\u0026ndash;76. https://doi.org/10.1016/B978-0-444-82033-4.50012-X\\u003c/li\\u003e\\n \\u003cli\\u003eOatley JM (2018) Recent advances for spermatogonial stem cell transplantation in livestock. Reprod Fertil Dev 30:44\\u0026ndash;49. https://doi.org/10.1071/RD17418\\u003c/li\\u003e\\n \\u003cli\\u003eOda A, Bailey KM, Lewbart GA, Griffith EH, Posner LP (2014) Physiologic and biochemical assessments of koi (\\u003cem\\u003eCyprinus carpio\\u003c/em\\u003e) following immersion in propofol. J Am Vet Med Assoc 245:1286\\u0026ndash;1291. https://doi.org/10.2460/javma.245.11.1286\\u003c/li\\u003e\\n \\u003cli\\u003eOkutsu T, Yano A, Nagasawa K, Shikina S, Kobayashi T, Takeuchi Y, Yoshizaki G (2006) Manipulation of fish germ cell: visualization, cryopreservation and transplantation. J Reprod Dev 52:685\\u0026ndash;693. https://doi.org/10.1262/jrd.18096\\u003c/li\\u003e\\n \\u003cli\\u003eOliveira DJ, Ashikaga FY, Foresti F, Senhorini JA (2017) Conservation status of the \\u0026ldquo;Piracanjuba\\u0026rdquo; \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e (Valenciennes, 1850) (Characiformes, Bryconidae): basis for management programs. Biodiversidade Brasileira 7:18\\u0026ndash;33. https://doi.org/10.37002/biodiversidadebrasileira.v7i1.612\\u003c/li\\u003e\\n \\u003cli\\u003eRivers N, Daly J, Jones R, Temple-Smith P (2020) Cryopreservation of testicular tissue from Murray River rainbowfish, \\u003cem\\u003eMelanotaenia fluviatilis\\u003c/em\\u003e. Sci Rep 10:19355. https://doi.org/10.1038/s41598-020-76378-7\\u003c/li\\u003e\\n \\u003cli\\u003eRosa I, Martinez E, Digmayer M, Doretto L, N\\u0026oacute;brega R (2023) Successful cryopreservation of spermatogonia stem cells of Neotropical catfish (Rhamdia quelen) and enriched germ cell transplantation into common carp (\\u003cem\\u003eCyprinus carpio\\u003c/em\\u003e) testes. Fishes 8:2\\u0026ndash;13. https://doi.org/10.3390/fishes8100478\\u003c/li\\u003e\\n \\u003cli\\u003eSchottger RA, Julin AM (1967) Efficacy of MS-222 as an anesthetic on four salmonids. Investigations in Fish Control. Bureau of Sport Fisheries and Wildlife 13:4\\u0026ndash;13. https://api.semanticscholar.org/CorpusID:71068834\\u003c/li\\u003e\\n \\u003cli\\u003eSiqueira-Silva DHD, Dos Santos Silva AP, da Silva Costa R, Senhorini JA, Ninhaus-Silveira A, Ver\\u0026iacute;ssimo-Silveira R (2021) Preliminary study on testicular germ cell isolation and transplantation in an endangered endemic species \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e (Characiformes: Characidae). Fish Physiol Biochem 47:767\\u0026ndash;776. https://doi.org/10.1007/s10695-019-00631-8\\u003c/li\\u003e\\n \\u003cli\\u003eSladky KK, Clarke EO III (2016) Fish surgery: presurgical preparation and common surgical procedures. Vet Clin North Am Exot Anim Pract 19:55\\u0026ndash;76. https://doi.org/10.1016/j.cvex.2015.08.008\\u003c/li\\u003e\\n \\u003cli\\u003eSwaminathan TR, Dharmaratnam A, Raja SA, Raj NS, Lal KK (2020) Establishment and cryopreservation of a cell line derived from caudal fin of endangered catfish \\u003cem\\u003eClarias dussumieri\\u003c/em\\u003e, Valenciennes, 1840. J Fish Biol 96:722\\u0026ndash;730. https://doi.org/10.1111/jfb.14265\\u003c/li\\u003e\\n \\u003cli\\u003eThelie A, Bailliard A, Seigneurin F, Zerjal T, Boichard M, Blesbois E (2019) Chicken semen cryopreservation and use for the restoration of rare genetic resources. Poult Sci 98:447\\u0026ndash;455. https://doi.org/10.3382/ps/pey360\\u003c/li\\u003e\\n \\u003cli\\u003eTonella LH, Dias RM, Vitorino OB, Fugi R, Agostinho AA (2019) Estado de conserva\\u0026ccedil;\\u0026atilde;o e bioecologia de \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e (Characiformes: Bryconidae), uma esp\\u0026eacute;cie de peixe end\\u0026ecirc;mica da bacia do rio Paran\\u0026aacute; (Brasil) amea\\u0026ccedil;ada de extin\\u0026ccedil;\\u0026atilde;o.\\u0026nbsp;Neotrop Ichthyol 17:e190030. https://doi.org/10.1590/1982-0224-20190030\\u003c/li\\u003e\\n \\u003cli\\u003eTort L (2011) Stress and immune modulation in fish. Dev Comp Immunol 35:1366\\u0026ndash;1374. https://doi.org/10.1016/j.dci.2011.07.002\\u003c/li\\u003e\\n \\u003cli\\u003eWildgoose WH (2000) Fish surgery: an overview. Fish Vet J 5:22\\u0026ndash;36. https://www.fishy.ru/fvsjournalissue5.pdf#page=3\\u003c/li\\u003e\\n \\u003cli\\u003eWylie MJ, Kitson J, Russell K, Yoshizaki G, Yazawa R, Steeves TE, Wellenreuther M (2023) Fish germ cell cryobanking and transplanting for conservation. Mol Ecol Resour 25:e13868. https://doi.org/10.1111/1755-0998.13868\\u003c/li\\u003e\\n \\u003cli\\u003eXie X, N\\u0026oacute;brega R, P\\u0026scaron;enička M (2020) Spermatogonial stem cells in fish: characterization, isolation, enrichment, and recent advances of in vitro culture systems. Biomolecules 10:644. https://doi.org/10.3390/biom1004\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Endangered species, gonadectomy, SSC, surgery, piracanjuba\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6681336/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6681336/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eSurgical techniques in aquatic species are underdeveloped, despite these species comprising a significant superclass among vertebrates. In the context of species preservation, studies involving the use of reproductive tissues typically involve the euthanasia of donors. Thus, in animals at high risk of extinction, the practice of sacrificing them presents a point of contradiction between ex situ conservation efforts. The objective of this study was to compare the viability of spermatogonia stem cells (SSCs) collected from \\u003cem\\u003eBrycon orbignyanus\\u003c/em\\u003e using conventional methods (euthanasia) versus a surgical procedure. Lateral celiotomy was performed on 27 immatures males to obtain a portion of gonadal tissue. The fish were divided into three groups (n\\u0026thinsp;=\\u0026thinsp;9), with each group receiving polyglactin 910, polyester, or catgut sutures, respectively, for celiorrhaphy. Dermorraphy was performed using nylon sutures in all groups. A. An additional euthanasia group consisted of nine animals exposed to 20 mg/L of propofol. The survival rate over a 48-hour period was 100% for the polyglactin 910 group and 77.7% for the polyester and catgut groups. Additionally, the viability of SSCs was similar between the euthanasia and surgical procedures. The lateral celiotomy technique is feasible for obtaining SSCs in fish with laterally compressed anatomy, such as \\u003cem\\u003eB. orbignyanus\\u003c/em\\u003e. Additionally, the technique allows the preservation of SSCs as a model for endangered fish species.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Celiotomy to collect spermatogonial stem cells in Brycon orbignyanus for species preservation\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-05-26 01:49:59\",\"doi\":\"10.21203/rs.3.rs-6681336/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"a9d57629-20a7-445d-821d-7c02ed39f0ca\",\"owner\":[],\"postedDate\":\"May 26th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-06-12T05:08:27+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-05-26 01:49:59\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6681336\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6681336\",\"identity\":\"rs-6681336\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}