Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species

Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species Lucia Suárez López, Paulo Sérgio Monzani, Gabriella Braga Carvalho, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4266695/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Fish Physiology and Biochemistry → Version 1 posted 8 You are reading this latest preprint version Abstract Piracanjuba ( Brycon orbignyanus ) is an endangered fish species from the Neotropical region. The establishment of a cryobank using spermatogonial stem cells (SSCs) and subsequent production of a germline chimera is thus a promising strategy for such species. In the present work, procedures for the isolation and cryopreservation of piracanjuba SSCs and subsequent transplantation into sterile recipients were established. The piracanjuba SSCs were obtained by Percoll density gradient centrifugation and differential plating. SSC fractions were evaluated by relative ddx4 expression, alkaline phosphatase activity, and light microscopy. SSC cryopreservation was performed using five cryoprotectants at three different concentrations. The mix of the cells from the 20% and 30% Percoll density gradients showed 58.35 ± 0.03% purity of SSCs. The purity of SSCs increased to 66.00 ± 0.01% after differential plating. The relative ddx4 expression was 3.5 times higher in cells from the Percoll density gradient centrifugation than in the gonad and cells after differential plating. Propanediol (1M) was the most effective cryoprotector evaluated ( P = 1.000 ), showing 90.75 ± 1.85% cell viability. Freshly isolated and cryopreserved cells from the Percoll density gradient centrifugation were transplanted into a sterile male adult triploid hybrid with germ cell-less gonads. SSCs were observed in the germinal epithelium of the testes of recipients 20 days after transplantation. The results are promising for obtaining functional germline chimeras in Neotropical fish. Consequently, the procedures established here can be applied in future actions for the conservation and reconstitution of the piracanjuba in case of extinction. conservation spermatogonia transplantation gene bank germline chimera Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Brycon orbignyanus ( Characidae, Bryconinae ), popularly known as piracanjuba, is a migratory fish species distributed in the basin formed by the Uruguay and Paraná rivers (Zaniboni Filho et al. 2006 ). Although this species has a consumer market (BORBA et al. 2006 ) and relevance for fisheries and aquaculture, the wild populations have declined, mainly affected by riparian deforestation and the fragmentation of rivers caused by hydroelectric plants (Agostinho, Â. A., Thomaz, S. M., & Gomes 2005). Such events contributed to its current status on the IUCN Red List classified as EN A2c (EN – endangered; A2c - ≥50% reduction in observed, estimated, inferred, or suspected population over the last ten years or three generations, where there was a decline in area of occupancy, extent of observation, and habitat quality or both) (ICMBio 2018 ). Strategies have been implemented to maintain the natural populations of piracanjuba, such as captive breeding and restocking programs (Rodriguez-Rodriguez et al. 2010 ). However, restocking requires one to maintain the genetic variability and prevent a deviation in the sex ratio. Captive-bred Piracanjuba produces mostly males, hindering in situ maintenance and reducing restocking effectiveness (Lopera-barrero 2007 ; Rodriguez-Rodriguez et al. 2010 ; Zardo et al. 2021 ). Long-term gene banking using cryopreserved samples is promising in the piracanjuba, especially using SSCs because a high percentage of males are available. In vitro cell preservation allows for subsequent transplantation use in the reconstitution of species by generating germline chimeras (xenogenesis) (Franěk et al. 2019 ; Marinović et al. 2019 ). This approach could be considered a solid strategy to conserve and reconstitute piracanjuba, preserving important genetic components (Cinalli et al. 2008 ). Despite using sperm in cryopreservation for its ease of management and cryopreservation (Viveiros et al. 2012 ); reconstitution via androgenesis is difficult to achieve and presents problems regarding survival, genetic variability, and sex ratio (Yasui et al. 2010 ). Cryopreservation protocols for fish embryos and oocytes have been unviable due to their large size, high yolk content, and high sensitivity to cooling (Robles et al. 2009 ; Labbé et al. 2013 ). Primordial germ cells, SSCs, and oogonial stem cells were successfully cryopreserved, transplanted, and used to produce a germline chimera for reconstitution. New individuals can be generated through cell transplantation using a sterile recipient to conserve endangered species (Hartung et al. 2014 ). Germ cell genetic banks were established in some fish species, such as Oncorhynchus mykiss (Okutsu et al. 2006 ), Danio rerio (Marinović et al. 2019 ), Tinca tinca (Linhartová et al. 2014 ; Marinović et al. 2017 ), and Oryzias latipes (Seki et al. 2017 ). Reconstitution through germ cell transplantation can be performed for blastula-stage embryos (Takeuchi et al. 2001 ), newly hatched larvae (Takeuchi et al. 2003 ), and adult animals (Lacerda et al. 2010 ). Regardless of the stage at which the germ cell transplant will be performed, recipients must be sterile and free of endogenous germ cells, which allows the proliferation and colonization of exogenous cells in the recipient’s gonad (Brinster et al. 2003 ; Lacerda, S.R. Batlouni, S.B.G. Silva, C.S.P. Homem 2006; Piva et al. 2018 ; Xu et al. 2019 ). Sterile recipients can be produced using techniques such as chromosome manipulation to generate triploids (Adamov et al. 2017 ; Do Nascimento et al. 2021 ), species hybridization (Piva et al. 2018 ; Xu et al. 2019 ), combination of both these methods (Piva et al. 2018 ; Xu et al. 2020 ), injection of morpholinos into embryos (dead end gene) (Franěk et al. 2022 ; Fujihara et al. 2022 ), as well as through high temperatures (Pandit et al. 2015 ; Nozu and Nakamura 2020 ) or busulfan chemical treatment associated with high temperatures (Lacerda et al. 2008 ; Majhi et al. 2017 ). Based on previous studies, cryopreservation and transplantation of germline cells are essential tools for the conservation of endangered species. This study aimed to establish procedures for isolating and cryopreserving SSCs from piracanjuba ( Brycon orbignyanus ). Additionally, the effectiveness of transplanting SSCs into the sterile testes of adult triploid hybrid recipients ( Astyanax altiparanae female X Astyanax fasciatus male) was evaluated. 2. Materials and methods 2.1 Ethical considerations for the use of animals Animal collection and experiments were carried out at the National Center for Research and Conservation of Continental Aquatic Biodiversity of the Chico Mendes Institute for Biodiversity Conservation (CEPTA/ICMBio) in Pirassununga – São Paulo, Brazil. The experiments were conducted with the approval of the Animal Ethics Committee from CEPTA. (CEUA/CEPTA #02031.000088/2021-61). 2.2 Histological analysis of the testes of piracanjuba (Brycon orbignyanus) To determine the appropriate time of SSC obtention from piracanjuba, testes were collected from sexually mature juveniles at 15 months (n = 2) and adults at 8 years old. Testes of juveniles were collected in March 2020 (resting period). For adults, they were collected (n = 2) at the height of the breeding season in December and in June (n = 2) in the winter season. Before test collection, the animal was euthanized using a solution of 200 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil). The collected gonads were fixed in Bouin fixative for 24 hours at room temperature and then transferred into 70% ethanol. Subsequently, the material was processed with conventional histology procedures, using a series of dehydration steps in increasing concentrations of ethanol (70%, 80%, 90%, and 100%), bleaching in xylene, and embedding in paraplast® (Sigma # SLBS8607, St. Louis, USA). 5 µm thick histological sections were obtained using a microtome equipped with a steel blade (Leica RM2235, Lincolnshire, USA). Slides were stained with hematoxylin and eosin, analyzed by microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and photographed (Nikon DSRi2, Nikon, Tokyo, Japan). The gonadal development spermatogenesis analyses were evaluated based on previous studies Brown-Peterson et al ( 2011 ). 2.3 Isolation of spermatogonial stem cells One male of piracanjuba ( Brycon orbignyanus ) collected in June weighing 1.345 kg and a standard length of 44 cm, was used for isolatation of the SSCs. The male was euthanized as mentioned in the previous section. The testis was collected, weighing 0.9858 g, and two small parts were separated: one was fixed in Bouin (Exodus Scientific #FB08835SO, Sumaré, Brazil) for 24 hours for further histological analysis; and the other part was stored at -80°C for gene expression analysis. Most of the testis was used to isolate SSCs through enzymatic dissociation and Percoll density gradient centrifugation (see below). The testis was transferred into a 90 × 15 mm sterile Petri dish containing Leibovitz cell culture medium (L-15, Gibco #21083027, Grand Island, USA) supplemented with 0.5% fetal bovine serum, 2 mM glutamine (Sigma #G7513, St. Louis, USA), 1 mM pyruvate (Sigma #S8636, St. Louis, USA), 1× MEM vitamin solution (Sigma #M6895 St. Louis, USA), 1× nonessential amino acid MEM solution (Sigma #M7147, St. Louis, USA), and 1× antibiotic and antimycotic solution (Sigma #A5955, St. Louis, USA). In a laminar flow cabinet (FUH12, VECO, Campinas - SP, Brazil), the testis was minced using a scalpel and washed three times with a supplemented Leibovitz (L-15) medium to remove excess blood cells. The minced testis was transferred to 15 mL centrifuge tubes, adding 10 mL of supplemented Leibovitz (L-15) medium for each 1 g of tissue and 2 mg/mL collagenase type I from Clostridium histolyticum (Sigma #C0130, St. Louis, USA). The centrifuge tube was placed in an orbital shaker at 60 r/min (PHOENIX, São Paulo, Brazil) and incubated for 2 hours at room temperature (~ 27°C). Then, 20 µg/mL DNase I (Sigma‒Aldrich #SLBT5559, St. Louis, USA) was added and incubated for an additional hour on a shaker. The cell suspension was filtered through a 50-µm nylon mesh to eliminate debris and transferred to a 15-mL tube. The final volume was adjusted to 10 mL with supplemented Leibovitz (L-15) medium and centrifuged at 300 x g for 5 minutes (Eppendorf 5702, Hamburg, Germany) at 25°C. The cell pellet was resuspended in 10 mL of supplemented Leibovitz (L-15) medium and centrifuged at 300 × g for 5 minutes to remove the excess collagenase and DNase. This step was performed twice. SSCs were fractionated using the Percoll density gradient centrifugation in concentrations of 10, 20, 30, and 40% Percoll® Plus (Sigma‒Aldrich # SLBH8181 V, St. Louis, USA). The Percoll density gradient centrifugation was performed in 15-mL tubes. The cell suspension was carefully pipetted on the surface of the 10% density gradient Percoll solution and centrifuged at 800 × g for 30 minutes at 25°C. The collected fractions were resuspended in 10 mL of supplemented Leibovitz (L-15) medium and centrifuged at 300 × g for 8 minutes at 25°C to remove the excess Percoll. The cells from the resultant cell pellet were suspended in 1 mL of supplemented Leibovitz (L-15) medium. Cell suspensions were analyzed by microscopy to assess the fractions containing the highest percentage of SSCs. The fraction from the 20% and 30% Percoll density gradients containing the highest concentrations of SSCs were combined, and 5 mL of D-MEM media culture was added (Dulbecco's Modified Eagle Medium, Gibco #31053028, Grand Island, USA). The cells were centrifuged twice at 300 × g for 5 minutes to remove the fetal bovine serum from the previous solution, and this permitted the labeling of stem cells with alkaline phosphatase, a marker of pluripotent stem cells and SSC (Hong et al. 1996, 2004 ). The cells were resuspended in 500 µL of D-MEM containing 1 µL of alkaline phosphatase substrate (Life Technologies #A14353, Frederick, USA). Fluorescent cells were verified by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and digital images were obtained (Nikon DSRi2, Nikon, Tokyo, Japan). After confirming the alkaline phosphatase activity of the SSCs, 10 µL of cell suspension was used to quantify the cell concentration using a hemocytometer. The viability of cells was assessed in triplicate using 30 µL of cell suspension and 5 µL of 0.4% trypan blue (Gibco #15250061, Grand Island, USA). Cell counting was performed using the method suggested by Louis and Siegel ( 2011 ). The cell suspension from the Percoll density gradient centrifugation was used for the differential plating. The culture media was composed of Dulbecco's modified Eagle’s medium (high glucose DMEM) (Gibco # 11965, Grand Island, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine (Sigma #G7513, St. Louis, USA), 1 mM pyruvate (Sigma #S8636, St. Louis, USA), 1× MEM vitamin solution (Sigma #M6895 St. Louis, USA), 1× MEM nonessential amino acid solution (Sigma #M7147, St. Louis, USA), and 1× antibiotic and antimycotic solution (Sigma #A5955, St. Louis, USA). The cells were cultured at a concentration of 1.5×10 7 SSCs/mL in a 90 x 15 mm sterile Petri dish with agar 1% for 14 h in an atmosphere of 5% CO 2 and 100% humidity at 30°C (Sanyo Electric, MCO-20AIC, Sakata, Japan). Then, the cells in suspension were recovered, centrifuged at 300 × g for 8 minutes at 25°C, and resuspended in supplemented Leibovitz (L-15) medium solution. The percentages of SSCs, viability, and alkaline phosphate activity were determined as described for the cells from the Percoll density gradient centrifugation step. These steps are illustrated in Fig. 1 . 2.4 Characterization of isolated spermatogonial stem cells The testis and cells from the purification steps, a mix of the bands from the 20% and 30% Percoll density gradient centrifugation and differential plating, were sampled. All samples were stored at -80°C until determination of the relative ddx4 expression, using β-actin as an endogenous gene. Ddx4 is considered a marker gene of SSCs. 2.5 Primer design for the gene of interest and endogenous gene Primers were designed from the alignment of the β-actin and ddx4 coding region sequences from different fish species deposited at the National Center for Biotechnology Information (NCBI). Astyanax mexicanus β-actin and ddx4 sequences were input into the Nucleotide BLAST program ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and sequences with more significant identities were selected primarily from the Characiforme order and after from others, such as Siluriformes and Cypriniformes. Coding regions were obtained by the Open Reading Frame Finder program ( https://www.ncbi.nlm.nih.gov/orffinder/ ). The coding regions were aligned using the Multalign program ( http://multalin.toulouse.inra.fr/multalin/ ). The conserved regions were preferentially selected for the design of specific primers by the Primer-BLAST program ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), using A. mexicanus sequences as a reference. 2.6 cDNA synthesis, primer specificity analysis, and sequencing Total RNA extraction from the samples was performed with TRIzol® Reagent (Ambion #15596026, Carlsbad, USA) following the manufacturer's instructions. RNA was quantified using a QIAxpert spectrophotometer (Qiagen, Hilden, Germany). Before cDNA synthesis, 1 µg of RNA from each sample was treated with DNAse I (Sigma‒Aldrich #SLBR4100 V, St. Louis, USA) and used for first-strand cDNA synthesis with a SuperScript III First-Strand Synthesis System kit (Invitrogen #18080051, Vilnius, Lithuania), according to the manufacturer instructions. The cDNA was used as a template for amplifying the regions specified by the β-actin and ddx4 primers by conventional PCR. PCR was performed by recombinant Taq DNA polymerase (Invitrogen #11615-010, Itapevi, Brazil) according to the manufacturer's instructions using 1 µL of cDNA from the samples and the corresponding designed primers. The cycling parameters used were 95°C for 5 minutes, followed by 35 cycles of amplification (95°C for 45 seconds, 55°C for 30 seconds, and 72°C for 30 seconds) with a final step at 72°C for 10 minutes. The reaction was performed in a Multigene thermocycler (Labnet International, Inc.). After amplification, the products were assessed for primer specificities by visualizing them on a 2% agarose gel. The recovered products were separated from the gel using the EZNA Gel Extraction Kit (OMEGA), cloned into the pGEM-T easy vector (Promega), and then sent for sequencing to verify the gene sequence. 2.7 Relative ddx4 expression analysis The relative ddx4 expression analysis was performed using β-actin as an endogenous gene. Standard curves for the genes of interest and endogenous genes were established using serial dilution (1:3) of a mixture of cDNA from all samples. Samples were diluted 1:5 for the gene expression analysis. A negative reaction control (NRC) was used for each gene. All samples were evaluated in triplicate; for each reaction, 10 µL of Quanti Nova SYBR Green PCR Master Mix (Qiagen #208054, Hilden, Germany), 8 µL of nuclease-free water, 1 µL of primer mix for target genes, and 1 µL of cDNA were used. The curves were made with each point in duplicate and showed efficiency between 0.9 and 1.1 and R ≅ of 0.99. The primers used in the qPCR are shown in Table 1 . Table 1 Sequences of primers used in qPCR for gene expression of piracanjuba SSCs ( Brycon orbygnianus ). Since b-actin is an endogenous gene and ddx4 is specific for spermatogonia. Genes Sequence (5´-3´) Amplicon size (bp) ß-actin F: 5´- CGTGCTGTCTTCCCATCCA-3´ 86 R: 5´- TCACCAACGTAGCTGTCCTTCTG-3´ ddx4 F: 5´- AAGACCACAGGAACTGAGCG-3´ 118 R: 5´- CCCGGTCTCCATGAATGCTT-3´ The amplification conditions were 95°C for 2 minutes, followed by 40 cycles of amplification (95°C for 5 seconds and 60°C for 15 seconds). The melting curve was performed from 60 to 95°C. The reaction was carried out in a Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany). Relative gene expression analysis was evaluated by the 2 −ΔΔCT method Larionov et al. ( 2005 ). 2.8 Cryopreservation of spermatogonial stem cells For the SSCs isolation and cryopreservation experiments, four adult males approximately four years old, weighing 739.29 ± 2.16 g, were used. Animals were euthanized using a solution of 200 mg/L eugenol. The testes collected had a mean weight of 5.13 ± 0.018 g. The purification of SSCs was carried out as described in the previous section “ Isolation of spermatogonial stem cells ”. The concentration of SSCs was determined microscopically using a hemocytometer. Before the cryopreservation trials, the cell viability of cultured cells was evaluated using Trypan Blue dye (Gibco #15250061, Grand Island, USA) (see above). Before freezing, the cell suspension was diluted 1:1 in cryo-solution using 50 µL of cell suspension at a concentration of 1.4×10 7 SSCs/mL and 50 µL of cryoprotectant solution. The cryoprotectant solutions were composed of 100 mM glucose and 0.5% bovine albumin serum (Inlab confidence # 1870, SP, Brazil), and the cryoprotectants ethylene glycol (Sigma # SHBB4592 V, St. Louis, USA), glycerol (Sigma # 72996TMV, St. Louis, USA), dimethylsulfoxide (Sigma # SHBG9653 V, St. Louis, USA), dimethylacetamide (Sigma # STBC4643 V, St. Louis, USA), and propanediol (Sigma # MKBF9063 V, St. Louis, USA). The cryoprotectant concentrations were 2 M, 3 M, and 4 M. The cell suspension and cryoprotectant solutions were diluted 1:1. The mixture of cryo-solutions and cells was loaded into 250-µL cryopreservation straws (IMV Technologies, France) with the total volume adjusted to 70 µL by cutting. The straws were sealed with polyvinyl alcohol (Sigma # 089K0037, St. Louis, USA), placed in cryotubes (Corning Incorporated # 430659, DF, Mexico), and transferred to a freezing container (CoolCell™ LX Freezing Container, BioCision). The freezing container containing the straws was transferred to an ultrafreezer at -80°C and kept for two hours (estimated period to reach − 80°C). The freezing rate was measured using a thermal recorder (OM-EL-USB-TC, Omega Instruments, Norwalk, USA) with a K-type thermocouple (OM-EL-USB-TC, Omega Instruments®, Norwalk, USA), which was inserted inside the straw. After freezing at -80°C, the cryotubes were immersed in liquid nitrogen at -196°C for storage, approximately for one week. Each sample was thawed in a water bath at 30°C for one minute. The straw was cut at the ends, and the cell suspension was transferred to a 1.5 mL centrifuge microtube and centrifuged at 350 x g for 5 minutes at 25°C (Hermle Labnet # Z326K, Wehingen, Germany). The supernatant was discarded, the pellet was resuspended in 30 µL of Hank’s solution, and 5 µL of Trypan Blue dye was added for subsequent viability analysis, which was performed in triplicates (see above). 2.9 Transplantation of spermatogonial stem cells Four eight-year-old male piracanjuba were used as cell donors for the transplantation of SSCs into adult triploid hybrid recipients. The animals were euthanized with 200 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil). The testes were collected and weighed, presenting an average of 0.43 ± 0.01 g. After isolation (see procedures above), SSCs from two donors were used for in vitro cryopreservation using the best cryoprotectant solution (Propanediol 1M). The SSCs were stained using the fluorescent membrane marker PKH26 (Sigma‒Aldrich #MKCK8658, St. Louis, USA). The cell pellet was suspended in 1 mL of supplemented L-15 medium, added to 8 µL of PKH26 dye, and incubated for 10 minutes. After this period, 1 mL of fetal bovine serum was added and incubated for one minute to inactivate the fluorescent marker. The volume of the cell suspension was adjusted to 10 mL with supplemented L-15 medium and centrifuged at 300 × g for 5 minutes. Three washes were performed with the same medium to eliminate excess dye and fetal bovine serum. Sterile male adult triploid hybrid recipients ( Astyanax altiparanae female X Astyanax fasciatus male) and germ cell-free gonads at 11 months of age were used for the transplantation of SSCs (Piva et al. 2018 ). A total of 24 animals were divided into two groups (n = 12). In the first group, transplantation was performed using freshly isolated SSCs from the Percoll density gradient centrifugation at a concentration of 2.3×10 6 SSCs/mL, resulting in 92% cell viability. In the second group, cryopreserved SSCs at 2.9×10 6 SSCs/mL with 88% cell viability from the Percoll density gradient were used in transplantation. An insulin syringe coupled with a tip was used to perform the transplant via the urogenital papilla. Approximately 70 µL of the cell suspension contained approximately 1.9×10 5 freshly isolated SSCs, and 2.4×10 5 cryopreserved SSCs were injected into each recipient. For transplantation, the recipients were anesthetized in a solution containing 100 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil). 2.10 Evaluation of chimerism by histochemistry The testes (n = 2) of transplanted recipients (freshly isolated SSCs and cryopreserved SSCs) at 10 and 20 days after transplant were collected and fixed in Bouin’s fixative (Exodus Científica #FB08835SO, Sumaré, Brazil) for 24 hours. The samples were dehydrated using increasing concentrations of ethanol (from 70–100%) and embedded in paraplast (Sigma # SLBS8607, St. Louis, USA). The paraffin blocks were cut at 5 µm using a microtome equipped with a steel blade (Leica RM2235, Lincolnshire, USA). The cut material was examined by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan) and photographed (NikonDSRi2, Nikon, Tokyo, Japan). The slides were visualized by the fluorescent marker PKH26 and counterstained with hematoxylin and eosin to identify cell types. 2.11 Statistical analysis Data were evaluated in triplicate for each cryoprotectant at different molar concentrations. Data were reported as the mean and standard error and analyzed for homogeneity using the Levene test (Brown and Forsythe 1974 ), and for normality, the Cramer‒von Mises test was used (Tamura 2008 ). Then, they were evaluated by ANOVA and Tukey’s test (α = 0.05). Statistical analyses were performed using STATISTICA 7.0 software. 3. Results 3.1 Histological characterization of the testis of piracanjuba The evaluated testes of the juvenile piracanjuba were small and translucent, presenting a gonadosomatic index (GSI) of 0.01 ± 0.00. The testes were identified in the development phase. Spermatogonia, a few cysts of spermatocysts, spermatids, and some spermatozoa isolated in the testis were observed in the germinal epithelium (Fig. 2 A-B). The testes of adult males collected in the winter were translucent and easily visualized in the abdominal cavity, presenting a GSI of 0.07 ± 0.00. A spermatogenesis in the regeneration phase was observed, showing a large number of spermatogonia and some cysts of spermatocysts, spermatids, and isolated spermatozoa in the testis (Fig. 2 C-D). The testes of adult males collected in the breeding season were large, firm, and had a blanched color, presenting a GSI of 0.86 ± 0.13. The spermatogenesis was active with the spermatozoa in the testicular lumen, where cysts of spermatocytes, spermatids, and a few spermatogonia were observed (Fig. 2 E-F). In addition, the animals released semen when slight manual pressure was applied to the coelomic cavity in the craniocaudal direction. 3.2 Isolation of spermatogonial stem cells The cell mix from the 20% and 30% bands of the Percoll density gradient centrifugation showed 58.35 ± 0.03% SSCs, 30.54 ± 0.02% spermatocytes, and 11.11 ± 0.05% sperm (Fig. 3 A). After differential plating, 66.00 ± 0.01% SSCs, 33.23 ± 0.01% spermatocytes, and 0.78 ± 0.02% sperm were obtained (Fig. 3 B). The cell viability of SSCs from the Percoll density gradient centrifugation was 92.94 ± 0.06%, increasing to 94.59 ± 0.13% after plating (Fig. 4 ). SSCs from the Percoll density gradient centrifugation and differential plating showed higher alkaline phosphatase activity than spermatocytes when visually compared to the intensity of fluorescence from both cells. This result indicates that the purified cells were SSCs (Fig. 5 ). 3.3 Analysis of piracanjuba gene sequences The sequencing of the amplified regions of the Brycon orbignyanus β-actin and ddx4 with 86 and 118 bp showed 99% and 92.37% identity, respectively, for Astyanax mexicanus sequences (NCBI Reference Sequence: XM_007247821.3; XM_022681259.1). Figures 6 A-B, show the alignments of the ddx4 and B-actin sequences from Astyanax mexicanus and B. orbignyanus . The products of the amplifications were visualized on a 2% agarose gel, showing the specificity of the primers (Fig. 6 C). 3.4 Relative ddx4 expression analysis The standard curves for the β-actin and ddx4 genes showed amplification efficiencies for the gene of interest and the endogenous control was as expected (0.9 to 1.1). The sampling points were located within the limits of the curves. The melting curve showed only one peak at a specific temperature for each amplification product, where no dimer formation was observed for the evaluated genes. The results of the relative ddx4 expression indicated that the expression of ddx4 was approximately 3.5 times higher in cells from the Percoll density gradient centrifugation (cell mix from the 20% and 30% bands) than in the testis and cells after differential plating (Fig. 7 ). This result suggests a decrease in the relative ddx4 expression in cultured cells from differential plating, even with higher SSC purity in differential plating. 3.5 Viability of spermatogonial stem cells after cryopreservation and freezing rate The freezing rate of each treatment is shown in Table 2 . The cooling rate was similar between the different cryoprotectant solutions, between a minimum of 6.33 ± 0.27 minutes (Etileglycol 1.5M) and a maximum of 9.67 ± 0.72 minutes (DMA 2M) (Table 3 ). While the exothermic peak was observed the solution without cryoprotectant had a lower exothermic peak with − 3.06 ± 0.62 (°C) and a time of 4.52 ± 0.30 (min) Table 2 . As expected, control group cells did not show cell viability after thawing without any cryoprotectant (P = 0.0000) (Table 3 ). For 1M propanediol, the cell viability was 94.74 ± 1.85% (P = 1.0000) after thawing (Table 3 ), this solution was considered the best cryoprotective solution. Followed by Propanediol 1.5M, 1M DMA and 2M DMSO presented a viability of 67.57 ± 8.15% (P = 0.2842) , 67.41 ± 12.85% (P = 0.0001) and 62.15 ± 6.45% (P = 1.0000) respectively after thawing (Table 3 ). Ethylene glycol had viability below 60% in the three molarities with values of 36.06 ± 4.18% (P = 0.9999) (1M), 35.35 ± 13.76% (P = 1.0000) (1.5M), and 45.66 ± 8.43% ( P = 0.9999 ) (2M) (Table 3 ). Table 2 Freezing rates of cryoprotectant solutions used in freezing spermatogonia stem cells of piracanjuba ( Brycon orbignyanus ) over two hours at -80°C. The cryoprotectant solutions were loaded in straws and frozen through slow cooling (approximately − 1°C/min). Data were obtained from three replicates and are expressed as the mean ± standard error. Cooling rate (-1°C/min) (20°C to 0°C) Freezing rate (-1°C/min) (0°C to -50°C) Freezing temperature Exoterm delay (min) Exoterm peak CONTROL 7.67 ± 0.72 35.67 ± 1.09 -0.50 ± 0.00 4.52 ± 0.30 -3.06 ± 0.62 PROPANEDIOL 1M 7.33 ± 0.27 28.00 ± 0.47 -3.83 ± 0.14 3.18 ± 0.16 -11.12 ± 1.02 PROPANEDIOL 1.5M 7.67 ± 0.54 31.33 ± 0.72 -5.83 ± 0.59 3.23 ± 0.09 -13.29 ± 1.86 PROPANEDIOL 2M 8.00 ± 0.47 29.00 ± 1.70 -8.33 ± 0.27 3.04 ± 0.11 -15.53 ± 1.28 DMA 1M 8.00 ± 0.82 29.67 ± 1.36 -3.50 ± 0.24 2.98 ± 0.06 -10.63 ± 1.14 DMA 1.5M 7.67 ± 0.54 31.00 ± 0.94 -6.83 ± 0.49 2.79 ± 0.10 -15.13 ± 1.15 DMA 2M 9.67 ± 0.72 32.67 ± 1.09 -9.67 ± 0.27 3.15 ± 0.10 -18.73 ± 1.09 DMSO 1M 7.33 ± 0.27 31.00 ± 0.94 -4.33 ± 0.27 3.18 ± 0.07 -11.40 ± 1.22 DMSO 1.5M 8.00 ± 0.94 31.33 ± 0.27 -5.50 ± 0.24 3.39 ± 0.20 -10.45 ± 1.45 DMSO 2M 8.67 ± 1.19 31.67 ± 1.19 -8.67 ± 0.54 3.04 ± 0.13 -15.57 ± 0.35 GLYCEROL 1M 7.33 ± 0.27 34.33 ± 1.19 -4.00 ± 0.24 2.84 ± 0.04 -10.92 ± 0.78 GLYCEROL 1.5M 7.00 ± 0.47 33.00 ± 0.47 -6.83 ± 1.11 3.11 ± 0.19 -15.34 ± 1.53 GLYCEROL 2M 6.67 ± 0.27 31.00 ± 0.82 -6.83 ± 0.36 3.31 ± 0.12 -15.76 ± 0.87 ETILEGLYCOL 1M 7.00 ± 0.00 30.00 ± 1.41 -3.67 ± 0.14 3.52 ± 0.18 -9.96 ± 0.50 ETILEGLYCOL 1.5M 6.33 ± 0.27 28.33 ± 0.72 -5.00 ± 0.24 3.12 ± 0.09 -11.37 ± 0.62 ETILEGLYCOL 2M 8.00 ± 0.82 31.00 ± 1.25 -8.17 ± 0.36 3.19 ± 0.08 -17.27 ± 1.16 Table 3 Viability of SSCs before freezing and post-thawing of piracanjuba ( Brycon orbignyanus ). Cell suspensions were diluted 1:1 in each cryo-solution, loaded in straws, and frozen through slow cooling (approximately − 1°C/min). Data were obtained from four replicates and are expressed as the mean ± standard error. Values with different letters in the same column indicate a significant difference. Treatment Viability (%) PRE-FREEZING CONTROL 87.68 ± 5.22 a PROPANEDIOL 1 M 90.74 ± 1.85 a 1.5 M 67.57 ± 8.15 ab 2 M 55.25 ± 12.41 abc DMA 1 M 67.41 ± 12.85 ab 1.5 M 40.99 ± 6.47 bcd 2 M 29.89 ± 4.04 bcd DMSO 1 M 34.85 ± 5.96 bcd 1.5 M 51.01 ± 10.94 abc 2 M 62.15 ± 6.45 abc GLYCEROL 1 M 52.49 ± 22.07 abc 1.5 M 21.73 ± 5.23 bcd 2 M 18.04 ± 6.5 cd ETILENOGLICOL 1 M 36.03 ± 4.18 bcd 1.5 M 35.35 ± 13.76 bcd 2 M 45.66 ± 8.43 abc POST-DEFROST CONTROL 0.00 ± 0.00 d P - value 0.0161 3.6 Evaluation of SSCs transplantation into the testis of adult triploid hybrid recipients In the testes of recipients transplanted (n = 2) with freshly isolated SSCs, PKH26-positive cells were visualized ten days after transplantation (Figs. 8 A and 8 B). Through counterstaining with hematoxylin and eosin, the initial development of the germinal epithelium was verified in the testes of the transplanted recipients, given that the triploid hybrids were germ cell-free testes (Figs. 8 C and 8 D). At 20 days after transplantation (n = 1) with freshly isolated and cryopreserved SSCs (n = 1), the initial spermatogenesis process was verified in the transplanted testes, observing the presence of SSCs and some spermatocytes (Fig. 9 A-D). Without transplantation, the individuals in the control group had testes without germ cells (Fig. 9 A). Some of the recipients' animals died in the days following the transplant (n = 3) for freshly isolated SSCs and cryopreserved SSCs. After a prolonged period of hormonal inductions, some of the transplanted animals were able to produce sperm, but the molecular analysis was inconclusive due to DNA degradation in the samples. The semen was used for in vitro fertilization of piracanjuba oocytes, as well as intracytoplasmic microinjection, but embryo development was not observed. In microscopy analysis, a few spermatozoids were found, but they were not activated. Other hormonal inductions were attempted, but they did not result in successful sperm production. The procedures were not included in the material and methods once the results were negative. 4. Discussion The evaluation of spermatogenesis stages in juveniles and adult piracanjuba collected in the winter and summer allowed for the identification of the adequate season and spermatogenesis stage to perform the isolation of SSCs. Adults collected in the winter were allowed to obtain a higher cellular concentration of purified SSCs for later use in cryopreservation or cell transplantation. The testes of juvenile animals were small, presenting a GSI of 0.01 ± 0.00, and even with SSCs, many animals are necessary to obtain an adequate quantity of cells to perform cryopreservation or transplantation in adult animals, as described in other studies (Majhi et al. 2014 ; Xu et al. 2019 ). Piracanjuba is an endangered species; therefore, the use of juveniles is unsuitable for the isolation of SSCs for cryopreservation or transplantation into adult recipients. In the absence of adults, however, testes from juveniles could be used in tissue cryopreservation and SSC isolation for transplantation in larvae (Marinović et al. 2019 ; Zhou et al. 2021 ). Adult testes collected in June were found in the regeneration stage of spermatogenesis, presenting a GSI of 0.07 ± 0.00. In this stage, SSCs were abundant in the gonads. Therefore, it is the most recommended site to isolate SSCs to reach high quantities of isolated SSCs. In the testes of adults collected in December, spermatozoa were observed in abundance in the lumen, ready to be released, and there was a low quantity of SSCs. Therefore, this stage of spermatogenesis is unfavorable for the isolation of SSCs; once the number of spermatozoa is more significant than SSCs, it makes cellular purification difficult. Studies involving the histological characterization and isolation of SSCs are essential to identify the moment the testes produce higher quantities of SSCs. Identifying this spermatogenesis stage is essential to obtain a higher concentration and better purification of SSCs, which can be used later in cell transplantation, cryopreservation, or in vitro cultivation (Poursaeid et al. 2020 ; Zupa et al. 2020 ). The regeneration spermatogenesis stage of adult piracanjuba was used to isolate SSCs. SSCs were isolated by enzymatic dissociation, Percoll density gradient centrifugation, and differential plating. This presented a degree of purity after differential plating of approximately 8% greater than the Percoll density gradient centrifugation, also obtaining superior viability. It was possible to eliminate mainly spermatocytes and sperm through differential plating, which was reduced from approximately 11% in the Percoll density gradient centrifugation to 0.78% after differential plating. Differential plating was applied as a purification step for the isolation of SSCs in other species, such as O. mykiss (Shikina et al. 2008 ), Oreochromis niloticus (Lacerda et al. 2014 ), Rhamdia quelen (Silva et al. 2016 ), and Prochilodus lineatus (Dias et al. 2020 ). Differential plating is considered an important step to increase the purity of germ stem cells during the purification step. The purification of SSCs is essential for the generation of high-quality in vitro genetic banks, which preserve the genetic material of endangered species, such as piracanjuba ( B. orbignyanus ). The relative ddx4 expression analysis of the testis and cells from the purification steps indicated that the Percoll density gradient centrifugation increased relative gene expression when compared with testis and differential plating. This result may be attributed to the increased concentration of SSCs obtained in the Percoll density gradient centrifugation (20% and 30% bands) compared to the whole testis. A higher percentage of SSCs was obtained in the differential plating step. However, the relative ddx4 expression was lower than that of cells from the Percoll density gradient centrifugation. This is because ddx4 is expressed in germline cells throughout all stages (Hartung et al. 2014 ). After differential plating, the expression was lower due to the loss of type B spermatogonia and spermatocytes. Ddx4 is mainly expressed in SSCs of O. niloticus , with slight expression observed in primary spermatocytes (Kobayashi et al. 2000 ). In Neotropical species like R. quelen and Colossoma macropomum , the ddx4 gene is primarily expressed in type A and B spermatogonia, with slight expression in spermatids and no expression in spermatozoa. In C. macropomum, ddx4 expression was reported in type A oogonia and oocytes, probably due to the accumulation of germplasm RNA (Ricci et al. 2018 ; Vasconcelos et al. 2019 ; Borella et al. 2020 ). In R. quelen , other genes showed high expression levels in cell fractions enriched by type A spermatogonia, such as plzf and pou5f3 (Lacerda et al. 2019 ). To identify SSCs in Percoll density gradient centrifugation and differential plating, we used alkaline phosphatase observing cells positive for this marker. Previously, alkaline phosphatase has been used to identify type A spermatogonia in vitro cultures of mice (Van Der Wee et al. 2001 ), as well as in fish such as O. latipes (Hong et al. 2004 ), Epinephelus coioides (Zhong et al. 2022 ) and Opsariichthys bidens (Chen et al. 2022 ). Cell cryopreservation is an essential strategy to preserve material with high genetic value. However, freezing is potentially fatal for most living cells, mainly due to the formation of intra- and extracellular ice crystals that lead to cell injury (Chowdhury and Ghosh 2021 ; Mahmud 2021 ). Furthermore, cells from different tissues or cells within the same tissue may have different rates of water efflux across the cell membrane; thus, the optimal cooling rate needs to be determined for each type of cell to be cryopreserved (Hoon et al. 2017 ). The cooling curve is an essential parameter for cell survival, and there is little information about the ideal cooling rate for SSCs (Redden et al. 2009 ). In this work, different cryoprotectants at different molarities were evaluated, as well as their respective freezing rates for cryopreservation of SSCs isolated from piracanjuba. The best cryoprotectant solution found was 1 M propanediol, presenting a viability of 90.74 ± 0.19 after thawing, a freezing temperature of -3.83 ± 0.14°C, a freezing rate of 28.00 ± 0.4 minutes, and an exothermic peak of -11 ± 1.02°C at 3.18 ± 0.16 minutes. Isolation and cryopreservation of SSCs have been successfully performed in various fish species. For instance, in R. quelen , 67% of cell viability was achieved after thawing by using MeOH 1.3 M (Rosa et al. 2023 ). Similarly, in T. tinca , the viabilities obtained were 57% using 1.5 M glycerol (Linhartová et al. 2014 ) and 50% at 3 M DMSO (Marinović et al. 2019 ). In a study by Patra et al. ( 2016 ), Labeo rohita had a 70% viability rate after being thawed using 1.4 M DMSO. Meanwhile, Zupa et al. ( 2020 ) found that Argyrosomus regius had a viability rate of 28.5% after being thawed using 10% de DMSO. The cryoprotectant and the molar concentration are species-specific; therefore, cryopreservation protocols are necessary for each species (Pšenička et al. 2016 ). The results obtained in this study were highly efficient for the cryopreservation of piracanjuba SSCs, reaching a high percentage of viability compared with the results of other studies. In addition, there are few cryopreservation protocols developed for SSCs of Neotropical fish species; this protocol shows promise for establishing future in vitro genetic banks. In the testes of adult triploid hybrid animals that received transplantation of freshly isolated and cryopreserved SSCs, the cells were visualized up to 20 days after transplantation by histological analysis. The combination of the two sterilization methods, hybridization between species and chromosomal manipulation, was efficient in producing recipient testes without germ cells, making them ideal recipients. Initial development of the germinal epithelium in the testes of the recipients indicated that the transplanted cells were proliferating, mainly due to the presence of spermatogonia and spermatocytes. The fact that cryopreserved germ cells colonize the recipient testes makes it possible to establish procedures that can be used in the reconstitution of piracanjuba in the event of extinction, as was done for other species (Yoshizaki and Lee 2018 ; Franěk et al. 2019 ). Some animals that received transplants were evaluated for semen production by hormonal induction. Despite obtaining sperm, in vitro fertilization and intracytoplasmic microinjection tests were unsuccessful. Additionally, it was not feasible to determine through molecular analysis whether the semen belonged to piracanjuba. This outcome created uncertainty about whether the sperm cells generated could have originated from the transplanted cells or if numerous hormonal stimulations enabled the receiving gonad to produce endogenous semen. Although it did not successfully obtain functional gamete production from the donor species, this work presents promising results for establishing procedures for the reconstitution of piracanjuba. The procedures performed are the first established for Neotropical species using adult triploid hybrid receptors. Transplantation of piracanjuba SSCs via urogenital papilla was previously performed using A. altiparanae male diploid recipients sterilized with busulfan and temperature techniques, producing endogenous and exogenous sperm (de Siqueira-Silva et al. 2019 ). In other studies, spermatogenesis was depleted using busulfan treatment, and SSC transplantation was performed, resulting in endogenous and exogenous spermatozoa production (Majhi et al. 2009 ; Lacerda et al. 2010 ). However, using 100% sterile animals, as in the case of triploid hybrids in which the testes are constituted only by support cells, is advantageous to these works cited since the competition for space and nutrients between endogenous and exogenous cells is eliminated (Dobrinski 2006 ). Therefore, some published works tested the production of recipients completely devoid of germ cells (Piva et al. 2018 ; Yoshikawa et al. 2018 ; Ponjarat et al. 2019 ). This strategy in some studies was successful in germ stem cell transplantation, where gametes were produced only from the donor species (Takeuchi et al. 2018 ; Xu et al. 2019 ). Conclusion In this study, effective procedures for gene banking were established for the first time in an endangered neotropical fish species, the piracanjuba ( B. orbignyanus ). These procedures include isolation, cryopreservation, and transplantation. The procedure was optimized by identifying the period of highest abundance of SSCs in the testis of adult animals. The purification methods which involved Percoll density gradient and differential plating have resulted in high purity and viability of SSCs. It has been observed that the SSCs fractioned have shown high alkaline phosphatase activity in comparison to spermatids and spermatocytes. The relative ddx4 expression analysis has indicated a decrease in cells from differential plating as compared to those from the Percoll density gradient. The best cryoprotectant solution for piracanjuba SSCs was a 1 M propanediol solution. Transplantation of freshly isolated and cryopreserved cells resulted in functional SSCs that developed the germinal epithelium in the testes of adult triploid hybrid recipients. Despite the initial evaluations suggesting low gamete production and no observed fertilization, further investigations are required to achieve functional gamete production. The gene banking procedures developed are innovative and have the potential for application in aquaculture and the conservation of endangered species like piracanjuba. Declarations Acknowledgments Authors are grateful to FAPESP (Young Investigator Award #2010/17429-1) which permitted the initial works in the yellowtail tetra and China Three Gorges Brasil (CTG Brasil, Project ANEEL #PD-00387-0418/2019) for the full funding of studies on the species. We also acknowledge CEPTA / ICMBio for kindly providing the facilities and experimental fish. Competing interests The authors declare that they have no competing interests. Funding The project was supported by the China Three Gorges Brasil (CTG Brasil) (Research & Development, Project ANEEL #PD-00387-0418/2019). Data Availability All data generated or analyzed during the current study are included in this published article. Ethics approval The experiments were conducted following the Animal Ethics Committee from the National Center for Research and Conservation of Continental Aquatic Biodiversity (CEUA/CEPTA # 02031.000088/2021-61). The authenticity of the document can be verified on the website https://sei.icmbio.gov.br/autenticidade informing the verification code: 9643544 and the code CRC: EC53598A . References Adamov NS de M, Nascimento NF do, Maciel ECS, et al (2017) Triploid Induction in the Yellowtail Tetra, Astyanax altiparanae, Using Temperature Shock: Tools for Conservation and Aquaculture. Journal of the World Aquaculture Society 48:741–750. https://doi.org/10.1111/jwas.12390 Agostinho, Â. A., Thomaz, S. M., & Gomes LC (2005) Conservação da biodiversidade em águas continentais do Brasil. Megadiversidade 1:70–78 BORBA MR, FRACALOSSI DM, PEZZATO LE (2006) Dietary energy requirement of piracanjuba fingerlings, Brycon orbignyanus, and relative utilization of dietary carbohydrate and lipid. Aquaculture Nutrition 12:183–191. https://doi.org/10.1111/j.1365-2095.2006.00401.x Borella MI, Chehade C, Costa FG, et al (2020) The brain-pituitary-gonad axis and the gametogenesis. In: Biology and Physiology of Freshwater Neotropical Fish. Elsevier, pp 315–341 Brinster CJ, Ryu BY, Avarbock MR, et al (2003) Restoration of fertility by germ cell transplantation requires effective recipient preparation. Biology of Reproduction 69:412–420. https://doi.org/10.1095/biolreprod.103.016519 Brown-Peterson NJ, Wyanski DM, Saborido-Rey F, et al (2011) A standardized terminology for describing reproductive development in fishes. Marine and Coastal Fisheries 3:52–70. https://doi.org/10.1080/19425120.2011.555724 Brown MB, Forsythe AB (1974) Robust tests for the equality of variances. Journal of the American Statistical Association 69:364–367. https://doi.org/10.1080/01621459.1974.10482955 Chen X, Kan Y, Zhong Y, et al (2022) Generation of a Normal Long-Term-Cultured Chinese Hook Snout Carp Spermatogonial Stem Cell Line Capable of Sperm Production In Vitro. Biology 11:. https://doi.org/10.3390/biology11071069 Chowdhury S, Ghosh S (2021) Cryopreservation of Stem Cells. In: Stem Cells. Springer Singapore, Singapore, pp 81–105 Cinalli RM, Rangan P, Lehmann R (2008) Germ Cells Are Forever. Cell 132:559–562. https://doi.org/10.1016/j.cell.2008.02.003 de Siqueira-Silva DH, dos Santos Silva AP, da Silva Costa R, et al (2019) Preliminary study on testicular germ cell isolation and transplantation in an endangered endemic species Brycon orbignyanus (Characiformes: Characidae). Fish Physiology and Biochemistry. https://doi.org/10.1007/s10695-019-00631-8 Dias GCM, Cassel M, Jesus LWO De, Borella MI (2020) Isolation , in vitro study , and stem cell markers for type A spermatogonia in a Characiformes species. 1–17. https://doi.org/10.1002/mrd.23394 Do Nascimento NF, Bertolini RM, Lopez LS, et al (2021) Heat-induced triploids in Brycon amazonicus: A strategic fish species for aquaculture and conservation. Zygote. https://doi.org/10.1017/S0967199421000125 Dobrinski I (2006) Transplantation of germ line stem cells for the study and manipulation of spermatogenesis. Ernst Schering Research Foundation workshop 175–193. https://doi.org/10.1007/3-540-31437-7_12 Franěk R, Cheng Y, Fučíková M, et al (2022) Who is the best surrogate for germ stem cell transplantation in fish? Aquaculture 549:. https://doi.org/10.1016/j.aquaculture.2021.737759 Franěk R, Marinović Z, Lujić J, et al (2019) Cryopreservation and transplantation of common carp spermatogonia. PLoS ONE 14:1–17. https://doi.org/10.1371/journal.pone.0205481 Fujihara R, Katayama N, Sadaie S, et al (2022) Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation. Marine Biotechnology 417–429. https://doi.org/10.1007/s10126-022-10128-w Hartung O, Forbes MM, Marlow FL (2014) Zebrafish vasa is required for germ-cell differentiation and maintenance. Molecular Reproduction and Development 81:946–961. https://doi.org/10.1002/mrd.22414 Hong Y, Liu T, Zhao H, et al (2004) Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proceedings of the National Academy of Sciences of the United States of America 101:8011–8016. https://doi.org/10.1073/pnas.0308668101 Hoon T, Park SC, Yang JH, et al (2017) Cryopreservation and its clinical applications. Integrative Medicine Research 6:12–18. https://doi.org/10.1016/j.imr.2016.12.001 ICMBio (2018) Livro Vermelho da Fauna Brasileira Ameaçada de Extinção. Brasília: ICMBio. Kobayashi T, Kajiura-Kobayashi H, Nagahama Y (2000) Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus. Mechanisms of Development 99:139–142. https://doi.org/10.1016/S0925-4773(00)00464-0 Labbé C, Robles V, Herraez MP (2013) Cryopreservation of gametes for aquaculture and alternative cell sources for genome preservation. Advances in Aquaculture Hatchery Technology 76–116. https://doi.org/10.1533/9780857097460.1.76 Lacerda, S.R. Batlouni, S.B.G. Silva, C.S.P. Homem LRF (2006) Germ cell type Days. In: Anim Reprod. p 2 Lacerda SM dos SN, Costa GMJ, de França LR (2014) Biology and identity of fish spermatogonial stem cell. General and Comparative Endocrinology 207:56–65. https://doi.org/10.1016/j.ygcen.2014.06.018 Lacerda SMSN, Batlouni SR, Assis LH, et al (2008) Germ cell transplantation in tilapia (Oreochromis niloticus). Cybium 32:115–118 Lacerda SMSN, Batlouni SR, Costa GMJ, et al (2010) A new and fast technique to generate offspring after germ cells transplantation in adult fish: The nile tilapia (Oreochromis niloticus) model. PLoS ONE 5:1–9. https://doi.org/10.1371/journal.pone.0010740 Lacerda SMSN, Martinez ERM, Mura ILDD, et al (2019) Duration of spermatogenesis and identification of spermatogonial stem cell markers in a Neotropical catfish, Jundiá (Rhamdia quelen). General and Comparative Endocrinology 273:249–259. https://doi.org/10.1016/j.ygcen.2018.10.018 Larionov A, Krause A, Miller WR (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinformatics 6:1–16. https://doi.org/10.1186/1471-2105-6-62 Linhartová Z, Rodina M, Guralp H, et al (2014) Isolation and cryopreservation of early stages of germ cells of tench (Tinca tinca). Czech Journal of Animal Science 59:381–390 Lopera-barrero NM (2007) Diversidade Genetica de Brycon orbignyanus. Universidade Estadual de Maringá Centro de Ciências Agrárias Louis KS, Siegel AC (2011) Cell Viability Analysis Using Trypan Blue: Manual and Automated Methods. In: Stoddart MJ (ed) Mammalian cell viability: methods and protocols. Humana Press, Totowa, NJ, pp 7–12 Mahmud N (2021) Cryopreservation of Stem Cell Product. In: Chandy M, Radhakrishnan VS, Sukumaran RK (eds). Springer International Publishing, Cham, pp 499–507 Majhi SK, Hattori RS, Rahman SM, Strüssmann CA (2014) Surrogate production of eggs and sperm by intrapapillary transplantation of germ cells in cytoablated adult fish. PLoS ONE 9:. https://doi.org/10.1371/journal.pone.0095294 Majhi SK, Hattori RS, Yokota M, et al (2009) Germ cell transplantation using sexually competent fish: An approach for rapid propagation of endangered and valuable germlines. PLoS ONE 4:1–8. https://doi.org/10.1371/journal.pone.0006132 Majhi SK, Rasal AR, Kushwaha B, Raizada S (2017) Heat and chemical treatments in adult Cyprinus carpio (Pisces cypriniformes) rapidly produce sterile gonads. Animal Reproduction Science 183:77–85. https://doi.org/10.1016/j.anireprosci.2017.05.015 Marinović Z, Li Q, Lujić J, et al (2019) Preservation of zebrafish genetic resources through testis cryopreservation and spermatogonia transplantation. Scientific Reports 9:1–10. https://doi.org/10.1038/s41598-019-50169-1 Marinović Z, Lujić J, Kása E, et al (2017) Cryosurvival of isolated testicular cells and testicular tissue of tench Tinca tinca and goldfish Carassius auratus following slow-rate freezing. General and Comparative Endocrinology 245:77–83. https://doi.org/10.1016/j.ygcen.2016.07.005 Nozu R, Nakamura M (2020) Influence of prolonged cultivation on sexual characteristics of sterilized female tilapia, Oreochromis mossambicus, induced by high-temperature treatment. Aquaculture 524:735245. https://doi.org/10.1016/j.aquaculture.2020.735245 Okutsu T, Suzuki K, Takeuchi Y, et al (2006) Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish. Proceedings of the National Academy of Sciences of the United States of America 103:2725–2729. https://doi.org/10.1073/pnas.0509218103 Pandit NP, Bhandari RK, Kobayashi Y, Nakamura M (2015) High temperature-induced sterility in the female Nile tilapia, Oreochromis niloticus. General and Comparative Endocrinology 213:110–117. https://doi.org/10.1016/j.ygcen.2015.01.028 Patra S, Mishra G, Dash SK, et al (2016) Transplantation worthiness of cryopreserved germ cells of Indian major carp rohu, Labeo rohita. Current Science 111:739–746. https://doi.org/10.18520/cs/v111/i4/739-746 Piva LH, de Siqueira-Silva DH, Goes CAG, et al (2018) Triploid or hybrid tetra: Which is the ideal sterile host for surrogate technology? Theriogenology 108:239–244. https://doi.org/10.1016/j.theriogenology.2017.12.013 Ponjarat J, Singchat W, Monkheang P, et al (2019) Evidence of dramatic sterility in F 1 male hybrid catfish [male Clarias gariepinus (Burchell, 1822) × female C. macrocephalus (Günther, 1864)] resulting from the failure of homologous chromosome pairing in meiosis I. Aquaculture 505:84–91. https://doi.org/10.1016/j.aquaculture.2019.02.035 Poursaeid S, Kalbassi MR, Hassani SN, Baharvand H (2020) Isolation, characterization, in vitro expansion and transplantation of Caspian trout (Salmo caspius) type a spermatogonia. General and Comparative Endocrinology 289:113341. https://doi.org/10.1016/j.ygcen.2019.113341 Pšenička M, Saito T, Rodina M, Dzyuba B (2016) Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells. Cryobiology 72:119–122. https://doi.org/10.1016/j.cryobiol.2016.02.005 Redden E, Davey R, Borjigin U, et al (2009) Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia. Cryobiology 58:190–195. https://doi.org/10.1016/j.cryobiol.2008.12.005 Ricci JMB, Martinez ERM, Butzge AJ, et al (2018) Characterization of vasa homolog in a neotropical catfish, Jundiá (Rhamdia quelen): Molecular cloning and expression analysis during embryonic and larval development. Gene 654:116–126. https://doi.org/10.1016/j.gene.2018.02.029 Robles V, Cabrita E, Paz Herráez M (2009) Germplasm cryobanking in zebrafish and other aquarium model species. Zebrafish 6:281–293. https://doi.org/10.1089/zeb.2009.0592 Rodriguez-Rodriguez M del P, Lopera-Barrero NM, Ribeiro RP, et al (2010) Diversidad genética de piracanjuba usada en programas de repoblación con marcadores microsatélites. Pesquisa Agropecuária Brasileira 45:56–63. https://doi.org/10.1590/s0100-204x2010000100008 Rosa IF, Martinez ERM, Digmayer M, et al (2023) Successful Cryopreservation of Spermatogonia Stem Cells of Neotropical Catfish (Rhamdia quelen) and Enriched Germ Cell Transplantation into Common Carp (Cyprinus carpio) Testes. Fishes 8:1–13. https://doi.org/10.3390/fishes8100478 Seki S, Kusano K, Lee S, et al (2017) Production of the medaka derived from vitrified whole testes by germ cell transplantation. Scientific Reports 7:1–11. https://doi.org/10.1038/srep43185 Shikina S, Ihara S, Yoshizaki G (2008) Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia. Molecular Reproduction and Development 75:529–537. https://doi.org/10.1002/mrd.20771 Silva MA, Costa GMJ, Lacerda SMSN, et al (2016) Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes. General and Comparative Endocrinology 230–231:48–56. https://doi.org/10.1016/j.ygcen.2016.03.012 Takeuchi Y, Yatabe T, Yoshikawa H, et al (2018) Production of functionally sterile triploid Nibe croaker Nibea mitsukurii induced by cold-shock treatment with special emphasis on triploid aptitude as surrogate broodstock. Aquaculture 494:45–56. https://doi.org/10.1016/j.aquaculture.2016.05.030 Takeuchi Y, Yoshizaki G, Takeuchi T (2001) Production of germ-line chimeras in rainbow trout by blastomere transplantation. Molecular Reproduction and Development 59:380–389. https://doi.org/10.1002/mrd.1044 Takeuchi Y, Yoshizaki G, Takeuchi T (2003) Generation of Live Fry from Intraperitoneally Transplanted Primordial Germ Cells in Rainbow Trout 1. 1149:1142–1149. https://doi.org/10.1095/biolreprod.103.017624 Tamura H (2008) 済無No Title No Title. Journal of Chemical Information and Modeling 53:287. https://doi.org/10.1017/CBO9781107415324.004 Van Der Wee KS, Johnson EW, Dirami G, et al (2001) Immunomagnetic isolation and long-term culture of mouse type a spermatogonia. Journal of Andrology 22:696–704. https://doi.org/10.1002/j.1939-4640.2001.tb02230.x Vasconcelos ACN, Streit DP, Octavera A, et al (2019) Isolation and characterization of a germ cell marker in teleost fish Colossoma macropomum. Gene 683:54–60. https://doi.org/10.1016/j.gene.2018.10.027 Viveiros ATM, Orfão LH, Nascimento AF, et al (2012) Effects of extenders, cryoprotectants and freezing methods on sperm quality of the threatened Brazilian freshwater fish pirapitinga-do-sul Brycon opalinus (Characiformes). Theriogenology 78:361–368. https://doi.org/10.1016/j.theriogenology.2012.02.015 Xu D, Yoshino T, De Bello Cioffi M, et al (2020) Production of donor-derived eggs after ovarian germ cell transplantation into the gonads of adult, germ cell-less, triploid hybrid fish. Biology of Reproduction 103:1289–1299. https://doi.org/10.1093/biolre/ioaa168 Xu D, Yoshino T, Konishi J, et al (2019) Germ cell-less hybrid fish: Ideal recipient for spermatogonial transplantation for the rapid production of donor-derived sperm. Biology of Reproduction 101:492–500. https://doi.org/10.1093/biolre/ioz045 Yasui GS, Fujimoto T, Arai K (2010) Restoration of the loach, Misgurnus anguillicaudatus, from cryopreserved diploid sperm and induced androgenesis. Aquaculture 308:S140–S144. https://doi.org/10.1016/j.aquaculture.2010.05.041 Yoshikawa H, Ino Y, Shigenaga K, et al (2018) Production of tiger puffer Takifugu rubripes from cryopreserved testicular germ cells using surrogate broodstock technology. Aquaculture 493:302–313. https://doi.org/10.1016/j.aquaculture.2018.05.016 Yoshizaki G, Lee S (2018) Production of live fish derived from frozen germ cells via germ cell transplantation. Stem Cell Research 29:103–110. https://doi.org/10.1016/j.scr.2018.03.015 Zaniboni Filho E, Reynalte-Tataje D, Weingartner M (2006) Potencialidad del género Brycon en la piscicultura brasileña. Revista Colombiana de Ciencias Pecuarias 19:233–240 Zardo ÉL, Fornari DC, Gioria J, et al (2021) Gonadal development period and sexual differentiation through histological analysis in Brycon orbignyanus (Valenciennes, 1850) (Characiformes: Bryconidae). Aquaculture 539:. https://doi.org/10.1016/j.aquaculture.2021.736636 Zhong C, Tao Y, Liu M, et al (2022) Establishment of a Spermatogonial Stem Cell Line with Potential of Meiosis in a Hermaphroditic Fish, Epinephelus coioides. Cells 11:. https://doi.org/10.3390/cells11182868 Zhou L, Wang X, Liu Q, et al (2021) Successful Spermatogonial Stem Cells Transplantation within Pleuronectiformes: First Breakthrough at inter-family Level in Marine Fish. International Journal of Biological Sciences 17:4426–4441. https://doi.org/10.7150/ijbs.63266 Zupa R, Martino NA, Marzano G, et al (2020) Meagre argyrosomus regius (Asso, 1801) stem spermatogonia: Histological characterization, immunostaining, in vitro proliferation, and cryopreservation. Animals 10:. https://doi.org/10.3390/ani10050851 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2024 Read the published version in Fish Physiology and Biochemistry → Version 1 posted Editorial decision: Revision requested 08 Jul, 2024 Reviews received at journal 02 May, 2024 Reviewers agreed at journal 28 Apr, 2024 Reviewers agreed at journal 23 Apr, 2024 Reviewers invited by journal 23 Apr, 2024 Editor assigned by journal 20 Apr, 2024 Submission checks completed at journal 17 Apr, 2024 First submitted to journal 14 Apr, 2024 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 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-4266695","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292325940,"identity":"3b2a73ba-3c28-49d0-831f-c616ac0fe3d2","order_by":0,"name":"Lucia Suárez López","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACPigtx8BMrBY2KG1MupbEBmJ1MLDxnzH8XFFRl97fzvzw4Y8KhmiDA4S0SOQYS545w5Y74zCbsTHPGYbcmYTsY5PgMZBsbOPJ3cDMwybN2MaQ20+Ew4x/Nv6TSDdg5mH/+fMfQ24bQS0MOWaSjQ0GCUAtbAy8DcTYIpFWZtlwLMEQ5BdpnmMShP3Cz394882Gmjp5/v7DDz/+qLHJ3XCAkDVoQIJE9aNgFIyCUTAKsAIA/Uc0elktgBoAAAAASUVORK5CYII=","orcid":"","institution":"Center for Scientific Research and Higher Education at Ensenada","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lucia","middleName":"Suárez","lastName":"López","suffix":""},{"id":292325941,"identity":"5e51b61d-7e0b-4995-b2d3-9091f796b717","order_by":1,"name":"Paulo Sérgio Monzani","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"Sérgio","lastName":"Monzani","suffix":""},{"id":292325942,"identity":"b46882ce-2b87-488d-ac36-4b9b01465804","order_by":2,"name":"Gabriella Braga Carvalho","email":"","orcid":"","institution":"Chico Mendes Institute of Biodiversity Conservation/National Center for Research and Conservation of Continental Aquatic Biodiversity","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gabriella","middleName":"Braga","lastName":"Carvalho","suffix":""},{"id":292325943,"identity":"8aea1ca2-ff9a-4918-9226-05f916e10ee6","order_by":3,"name":"Diógenes Henrique Siqueira Silva","email":"","orcid":"","institution":"Federal University of the South and Southeast of Pará","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Diógenes","middleName":"Henrique Siqueira","lastName":"Silva","suffix":""},{"id":292325944,"identity":"cf0f2fd0-0fd6-47b4-baf4-2ad0b1733156","order_by":4,"name":"Norberto Castro Vianna","email":"","orcid":"","institution":"China Three Gorges Corporation (CTG)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Norberto","middleName":"Castro","lastName":"Vianna","suffix":""},{"id":292325945,"identity":"56a9876f-39be-4647-b417-ba7239730bf2","order_by":5,"name":"George Shigueki Yasui","email":"","orcid":"","institution":"Chico Mendes Institute of Biodiversity Conservation/National Center for Research and Conservation of Continental Aquatic Biodiversity","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"George","middleName":"Shigueki","lastName":"Yasui","suffix":""},{"id":292325946,"identity":"b136957e-c169-43f5-8303-8272d4f709b2","order_by":6,"name":"José Augusto Senhorini","email":"","orcid":"","institution":"Chico Mendes Institute of Biodiversity Conservation/National Center for Research and Conservation of Continental Aquatic Biodiversity","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"José","middleName":"Augusto","lastName":"Senhorini","suffix":""}],"badges":[],"createdAt":"2024-04-15 02:01:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4266695/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4266695/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10695-024-01406-6","type":"published","date":"2024-09-27T15:57:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55322666,"identity":"325cc8fa-b2c6-4a5e-b695-fe4bbb0adc9c","added_by":"auto","created_at":"2024-04-25 16:28:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":160064,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic design of the steps for the enzymatic dissociation and isolation process of piracanjuba (\u003cem\u003eBrycon orbignyanus\u003c/em\u003e) spermatogonial cells. \u003cstrong\u003eA)\u003c/strong\u003e An adult male piracanjuba specimen was euthanized, and the testes were collected. \u003cstrong\u003eB)\u003c/strong\u003e Then, the testes were sliced into small fragments and subjected to the process of enzymatic dissociation using collagenase type I. \u003cstrong\u003eC)\u003c/strong\u003e After the enzymatic dissociation process, the SSCs were isolated using a Percoll density gradient (10%, 20%, 30%, and 40%). \u003cstrong\u003eD)\u003c/strong\u003eThe bands containing the highest number of SSCs (20% and 30%) were subjected to cell viability testing using the trypan blue membrane marker. \u003cstrong\u003eE)\u003c/strong\u003eAlkaline phosphatase activity was also tested for the confirmation of SSCs. \u003cstrong\u003eF)\u003c/strong\u003eFor better purification of the SSCs, the cells were subjected to differential plating for 14 hours, and after differential plating, the cell viability \u003cstrong\u003e(D)\u003c/strong\u003eand alkaline phosphatase activity \u003cstrong\u003e(E)\u003c/strong\u003e were verified again.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/96cd1f0eb5b64ef8908371a5.png"},{"id":55322693,"identity":"e1baa12e-d7ed-4261-a2ac-8d55691b53a8","added_by":"auto","created_at":"2024-04-25 16:28:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13991097,"visible":true,"origin":"","legend":"\u003cp\u003eTestes of juvenile (15-months old) and adult piracanjuba (Brycon orbignyanus) collected from November to December (spawning season) and from May to June (resting).\u003cstrong\u003e A)\u003c/strong\u003e Juvenile testes in the initial development phase, observing cysts of spermatogonia, spermatocytes, spermatids, and some spermatozoa. \u003cstrong\u003eB)\u003c/strong\u003e Higher magnification image of the dotted area in 2A, clearly identifying spermatogonial cysts, some spermatocytes, spermatids, and spermatozoa. \u003cstrong\u003eC) \u003c/strong\u003eTestis of an adult male collected between late May and early June, showing spermatogenesis in the regeneration phase, spermatogonia in proliferation in the testis and some spermatocytes, spermatids, and some residual spermatozoa can be observed.\u003cstrong\u003e D) \u003c/strong\u003eHigher magnification image of the dotted area in Figure 2C for better identification of spermatocytes and sperm.\u003cstrong\u003e E)\u003c/strong\u003e Testis collected from November to December in the mature phase with abundant spermatozoa in the lumen, while few spermatogonia were observed in the germinal epithelium. \u003cstrong\u003eF)\u003c/strong\u003eHigher magnification image of Figure 2E for identification of spermatogonia, spermatocytes, and spermatid cysts. Sg: spermatogonia, Sp: spermatocytes, St: spermatids, Sz: sperm, L: testicular lumen. \u003cstrong\u003eScale bar:\u003c/strong\u003e 200 µm (A, C, E)- 50 µm (B, D, F).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/509e099abb04d85ad22a9d24.png"},{"id":55322690,"identity":"4a1e5d9b-be6b-49a1-b90a-609b5680f033","added_by":"auto","created_at":"2024-04-25 16:28:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1195230,"visible":true,"origin":"","legend":"\u003cp\u003eSpermatogonia of piracanjuba (Brycon orbignyanus) isolated by enzymatic dissociation followed by Percoll gradient (mixture of 20 and 30% percoll gradients). \u003cstrong\u003eA)\u003c/strong\u003e Cells from the Percoll gradient. \u003cstrong\u003eB)\u003c/strong\u003e Cells from differential plating. SG: spermatogonia, SP: spermatocytes, SZ: sperm.\u003cstrong\u003eScale bar:\u003c/strong\u003e 50 µm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/da60c774a0f22cc3520210ed.png"},{"id":55322689,"identity":"2c3b7a19-c307-4745-b737-53f2ebec0f03","added_by":"auto","created_at":"2024-04-25 16:28:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":399665,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability analysis of spermatogonia of piracanjuba (Brycon orbignyanus) that were damaged during the process of enzymatic dissociation and isolated with Percoll gradient (mixture of 20 and 30% percoll gradients) using the trypan blue membrane marker. White arrows indicate viable cells where trypan blue could not penetrate the cell membrane. Black arrows indicate nonviable cells where trypan blue crosses the cell membrane. \u003cstrong\u003eScale bar:\u003c/strong\u003e 50 µm.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/2fe56b1296d6e9c722cddf3a.png"},{"id":55322688,"identity":"7d4f5c90-2fe4-4927-bc80-90603b84c37e","added_by":"auto","created_at":"2024-04-25 16:28:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":487608,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative image of alkaline phosphatase activity in SSCs of piracanjuba\u003cem\u003e \u003c/em\u003e(\u003cem\u003eBrycon orbignyanus\u003c/em\u003e) from differential plating. \u003cstrong\u003eA)\u003c/strong\u003eWhite arrows with a black outline indicate spermatogonia observed by light microscopy; \u003cstrong\u003eB)\u003c/strong\u003e Counterphoto of the image (\u003cstrong\u003eA)\u003c/strong\u003e whitearrows point to SSCs stained with fluorescent dye. \u003cstrong\u003eScale bar:\u003c/strong\u003e50 μm.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/d944def994b0037a49b4d707.png"},{"id":55323185,"identity":"dd2bc942-a56d-47fa-a725-e36f04afca31","added_by":"auto","created_at":"2024-04-25 16:36:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":156556,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment of the amplified region of the \u003cem\u003eddx4\u003c/em\u003e and \u003cem\u003eB-actin\u003c/em\u003egene of \u003cem\u003eAstyanax mexicanus\u003c/em\u003e and \u003cem\u003eBrycon orbignyanus\u003c/em\u003e. \u003cstrong\u003eA)\u003c/strong\u003e \u003cem\u003eddx4\u003c/em\u003e. \u003cstrong\u003eB)\u003c/strong\u003e \u003cem\u003eB-actin\u003c/em\u003e. NCBI Reference Sequence: XM_007247821.3; XM_022681259.1). \u003cstrong\u003eC) \u003c/strong\u003eEvaluation of primers designed on cDNA samples from gonads of \u003cem\u003eBrycon orbignyanus\u003c/em\u003e in 2% agarose showing the specificity of the primers. \u003cstrong\u003eL:\u003c/strong\u003e Ladder 1 kb plus (Invitrogen); \u003cstrong\u003e1:\u003c/strong\u003e \u003cem\u003eB-actin\u003c/em\u003e; \u003cstrong\u003e2:\u003c/strong\u003e \u003cem\u003eddx4\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/1a9d510c371dfe4e6e2114bb.png"},{"id":55323186,"identity":"8a02feb3-835b-432a-a093-3eb404df6ff0","added_by":"auto","created_at":"2024-04-25 16:36:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17215,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of piracanjuba\u003cem\u003e \u003c/em\u003e(\u003cem\u003eBrycon orbignyanus\u003c/em\u003e) \u003cem\u003eddx4\u003c/em\u003e gene for testis, SSCs isolated\u003cem\u003e \u003c/em\u003efrom\u003cem\u003e \u003c/em\u003ePercoll gradient\u003cem\u003e \u003c/em\u003e(mixture of 20 and 30%), and SSCs subjected to differential plating. Gonad (1); Percoll (3.623008), and differential plating (1.036733).\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/e528307fb54b6d4c1267cbc3.png"},{"id":55322692,"identity":"bc90f972-fb3f-4bf1-bba2-4850c87594bb","added_by":"auto","created_at":"2024-04-25 16:28:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1909949,"visible":true,"origin":"","legend":"\u003cp\u003eTestes of adult triploid hybrids analyzed by light microscopy ten days after transplantation. \u003cstrong\u003eA)\u003c/strong\u003e Analysis by fluorescence microscopy, where the white dotted lines indicate regions of spermatogonia marked with PKH26. \u003cstrong\u003eB)\u003c/strong\u003e Dotted lines indicate the region in\u003cstrong\u003e A \u003c/strong\u003ewhere PKH26 positive cells were identified. \u003cstrong\u003eC)\u003c/strong\u003e Testes of the control group without germ cells in the presence of testicular lumen (L) and interstitium (I). \u003cstrong\u003eD)\u003c/strong\u003e Transplanted testes with arrows indicating germinal epithelial cells (Ep) and macrophages (Mc). \u003cstrong\u003eScale bars:\u003c/strong\u003e A-B:100 µm; C: 200 µm; D: 50 µm.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/c134bdccad7f62a61563bcaa.png"},{"id":55322695,"identity":"768d5166-42c2-4cf1-bf42-097510a3159a","added_by":"auto","created_at":"2024-04-25 16:28:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1878396,"visible":true,"origin":"","legend":"\u003cp\u003eHistological section of testes of adult triploid hybrids analyzed by light microscopy 20 days after transplantation, where the slides were stained with hematoxylin and eosin. \u003cstrong\u003eA)\u003c/strong\u003e Testes of the control group presenting only testicular lumen and interstitium. \u003cstrong\u003eB) \u003c/strong\u003eRepresentative testes of a newly isolated spermatogonia host, where arrows indicate SSCs in the process of proliferation \u003cstrong\u003eC)\u003c/strong\u003e Testes of a recipient transplanted with cryopreserved cells, observing the proliferation and colonization of the testes. \u003cstrong\u003eD)\u003c/strong\u003e Higher magnification image of the \u003cstrong\u003eC\u003c/strong\u003e region, where spermatogonia and spermatocytes were identified in the recipient testes. Sg: spermatogonia; Sp: spermatocytes; I: interstitium; L: testicular lumen. \u003cstrong\u003eScale bars:\u003c/strong\u003eA-C: 200 µm; B-D: 50 µm.\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/52cadd6a555ff60d37486b5a.png"},{"id":65628081,"identity":"9815afc6-26b3-4745-b6ba-f61aac906918","added_by":"auto","created_at":"2024-09-30 16:17:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34511362,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4266695/v1/1500a20f-c670-43ed-9341-69ca9e4c4d8a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eBrycon orbignyanus\u003c/em\u003e (\u003cem\u003eCharacidae, Bryconinae\u003c/em\u003e), popularly known as piracanjuba, is a migratory fish species distributed in the basin formed by the Uruguay and Paran\u0026aacute; rivers (Zaniboni Filho et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Although this species has a consumer market (BORBA et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and relevance for fisheries and aquaculture, the wild populations have declined, mainly affected by riparian deforestation and the fragmentation of rivers caused by hydroelectric plants (Agostinho, \u0026Acirc;. A., Thomaz, S. M., \u0026amp; Gomes 2005). Such events contributed to its current status on the IUCN Red List classified as EN A2c (EN \u0026ndash; endangered; A2c - \u0026ge;50% reduction in observed, estimated, inferred, or suspected population over the last ten years or three generations, where there was a decline in area of occupancy, extent of observation, and habitat quality or both) (ICMBio \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Strategies have been implemented to maintain the natural populations of piracanjuba, such as captive breeding and restocking programs (Rodriguez-Rodriguez et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, restocking requires one to maintain the genetic variability and prevent a deviation in the sex ratio. Captive-bred Piracanjuba produces mostly males, hindering \u003cem\u003ein situ\u003c/em\u003e maintenance and reducing restocking effectiveness (Lopera-barrero \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rodriguez-Rodriguez et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zardo et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLong-term gene banking using cryopreserved samples is promising in the piracanjuba, especially using SSCs because a high percentage of males are available. \u003cem\u003eIn vitro\u003c/em\u003e cell preservation allows for subsequent transplantation use in the reconstitution of species by generating germline chimeras (xenogenesis) (Franěk et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Marinović et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This approach could be considered a solid strategy to conserve and reconstitute piracanjuba, preserving important genetic components (Cinalli et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite using sperm in cryopreservation for its ease of management and cryopreservation (Viveiros et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e); reconstitution via androgenesis is difficult to achieve and presents problems regarding survival, genetic variability, and sex ratio (Yasui et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Cryopreservation protocols for fish embryos and oocytes have been unviable due to their large size, high yolk content, and high sensitivity to cooling (Robles et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Labb\u0026eacute; et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrimordial germ cells, SSCs, and oogonial stem cells were successfully cryopreserved, transplanted, and used to produce a germline chimera for reconstitution. New individuals can be generated through cell transplantation using a sterile recipient to conserve endangered species (Hartung et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Germ cell genetic banks were established in some fish species, such as \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e (Okutsu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), \u003cem\u003eDanio rerio\u003c/em\u003e (Marinović et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eTinca tinca\u003c/em\u003e (Linhartov\u0026aacute; et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Marinović et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and \u003cem\u003eOryzias latipes\u003c/em\u003e (Seki et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Reconstitution through germ cell transplantation can be performed for blastula-stage embryos (Takeuchi et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), newly hatched larvae (Takeuchi et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and adult animals (Lacerda et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Regardless of the stage at which the germ cell transplant will be performed, recipients must be sterile and free of endogenous germ cells, which allows the proliferation and colonization of exogenous cells in the recipient\u0026rsquo;s gonad (Brinster et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lacerda, S.R. Batlouni, S.B.G. Silva, C.S.P. Homem 2006; Piva et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Sterile recipients can be produced using techniques such as chromosome manipulation to generate triploids (Adamov et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Do Nascimento et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), species hybridization (Piva et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), combination of both these methods (Piva et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), injection of morpholinos into embryos (dead end gene) (Franěk et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Fujihara et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), as well as through high temperatures (Pandit et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nozu and Nakamura \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) or busulfan chemical treatment associated with high temperatures (Lacerda et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Majhi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Based on previous studies, cryopreservation and transplantation of germline cells are essential tools for the conservation of endangered species.\u003c/p\u003e \u003cp\u003eThis study aimed to establish procedures for isolating and cryopreserving SSCs from piracanjuba (\u003cem\u003eBrycon orbignyanus\u003c/em\u003e). Additionally, the effectiveness of transplanting SSCs into the sterile testes of adult triploid hybrid recipients (\u003cem\u003eAstyanax altiparanae\u003c/em\u003e female X \u003cem\u003eAstyanax fasciatus\u003c/em\u003e male) was evaluated.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethical considerations for the use of animals\u003c/h2\u003e \u003cp\u003eAnimal collection and experiments were carried out at the National Center for Research and Conservation of Continental Aquatic Biodiversity of the Chico Mendes Institute for Biodiversity Conservation (CEPTA/ICMBio) in Pirassununga \u0026ndash; S\u0026atilde;o Paulo, Brazil. The experiments were conducted with the approval of the Animal Ethics Committee from CEPTA. (CEUA/CEPTA #02031.000088/2021-61).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Histological analysis of the testes of piracanjuba (Brycon orbignyanus)\u003c/h2\u003e \u003cp\u003eTo determine the appropriate time of SSC obtention from piracanjuba, testes were collected from sexually mature juveniles at 15 months (n\u0026thinsp;=\u0026thinsp;2) and adults at 8 years old. Testes of juveniles were collected in March 2020 (resting period). For adults, they were collected (n\u0026thinsp;=\u0026thinsp;2) at the height of the breeding season in December and in June (n\u0026thinsp;=\u0026thinsp;2) in the winter season. Before test collection, the animal was euthanized using a solution of 200 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil). The collected gonads were fixed in Bouin fixative for 24 hours at room temperature and then transferred into 70% ethanol. Subsequently, the material was processed with conventional histology procedures, using a series of dehydration steps in increasing concentrations of ethanol (70%, 80%, 90%, and 100%), bleaching in xylene, and embedding in paraplast\u0026reg; (Sigma # SLBS8607, St. Louis, USA). 5 \u0026micro;m thick histological sections were obtained using a microtome equipped with a steel blade (Leica RM2235, Lincolnshire, USA). Slides were stained with hematoxylin and eosin, analyzed by microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and photographed (Nikon DSRi2, Nikon, Tokyo, Japan). The gonadal development spermatogenesis analyses were evaluated based on previous studies Brown-Peterson et al (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Isolation of spermatogonial stem cells\u003c/h2\u003e \u003cp\u003eOne male of piracanjuba (\u003cem\u003eBrycon orbignyanus\u003c/em\u003e) collected in June weighing 1.345 kg and a standard length of 44 cm, was used for isolatation of the SSCs. The male was euthanized as mentioned in the previous section. The testis was collected, weighing 0.9858 g, and two small parts were separated: one was fixed in Bouin (Exodus Scientific #FB08835SO, Sumar\u0026eacute;, Brazil) for 24 hours for further histological analysis; and the other part was stored at -80\u0026deg;C for gene expression analysis. Most of the testis was used to isolate SSCs through enzymatic dissociation and Percoll density gradient centrifugation (see below).\u003c/p\u003e \u003cp\u003eThe testis was transferred into a 90 \u0026times; 15 mm sterile Petri dish containing Leibovitz cell culture medium (L-15, Gibco #21083027, Grand Island, USA) supplemented with 0.5% fetal bovine serum, 2 mM glutamine (Sigma #G7513, St. Louis, USA), 1 mM pyruvate (Sigma #S8636, St. Louis, USA), 1\u0026times; MEM vitamin solution (Sigma #M6895 St. Louis, USA), 1\u0026times; nonessential amino acid MEM solution (Sigma #M7147, St. Louis, USA), and 1\u0026times; antibiotic and antimycotic solution (Sigma #A5955, St. Louis, USA). In a laminar flow cabinet (FUH12, VECO, Campinas - SP, Brazil), the testis was minced using a scalpel and washed three times with a supplemented Leibovitz (L-15) medium to remove excess blood cells. The minced testis was transferred to 15 mL centrifuge tubes, adding 10 mL of supplemented Leibovitz (L-15) medium for each 1 g of tissue and 2 mg/mL collagenase type I from \u003cem\u003eClostridium histolyticum\u003c/em\u003e (Sigma #C0130, St. Louis, USA). The centrifuge tube was placed in an orbital shaker at 60 r/min (PHOENIX, S\u0026atilde;o Paulo, Brazil) and incubated for 2 hours at room temperature (~\u0026thinsp;27\u0026deg;C). Then, 20 \u0026micro;g/mL DNase I (Sigma‒Aldrich #SLBT5559, St. Louis, USA) was added and incubated for an additional hour on a shaker. The cell suspension was filtered through a 50-\u0026micro;m nylon mesh to eliminate debris and transferred to a 15-mL tube. The final volume was adjusted to 10 mL with supplemented Leibovitz (L-15) medium and centrifuged at 300 x g for 5 minutes (Eppendorf 5702, Hamburg, Germany) at 25\u0026deg;C. The cell pellet was resuspended in 10 mL of supplemented Leibovitz (L-15) medium and centrifuged at 300 \u0026times; g for 5 minutes to remove the excess collagenase and DNase. This step was performed twice.\u003c/p\u003e \u003cp\u003eSSCs were fractionated using the Percoll density gradient centrifugation in concentrations of 10, 20, 30, and 40% Percoll\u0026reg; Plus (Sigma‒Aldrich # SLBH8181 V, St. Louis, USA). The Percoll density gradient centrifugation was performed in 15-mL tubes. The cell suspension was carefully pipetted on the surface of the 10% density gradient Percoll solution and centrifuged at 800 \u0026times; g for 30 minutes at 25\u0026deg;C. The collected fractions were resuspended in 10 mL of supplemented Leibovitz (L-15) medium and centrifuged at 300 \u0026times; g for 8 minutes at 25\u0026deg;C to remove the excess Percoll. The cells from the resultant cell pellet were suspended in 1 mL of supplemented Leibovitz (L-15) medium. Cell suspensions were analyzed by microscopy to assess the fractions containing the highest percentage of SSCs.\u003c/p\u003e \u003cp\u003eThe fraction from the 20% and 30% Percoll density gradients containing the highest concentrations of SSCs were combined, and 5 mL of D-MEM media culture was added (Dulbecco's Modified Eagle Medium, Gibco #31053028, Grand Island, USA). The cells were centrifuged twice at 300 \u0026times; g for 5 minutes to remove the fetal bovine serum from the previous solution, and this permitted the labeling of stem cells with alkaline phosphatase, a marker of pluripotent stem cells and SSC (Hong et al. 1996, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The cells were resuspended in 500 \u0026micro;L of D-MEM containing 1 \u0026micro;L of alkaline phosphatase substrate (Life Technologies #A14353, Frederick, USA). Fluorescent cells were verified by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and digital images were obtained (Nikon DSRi2, Nikon, Tokyo, Japan). After confirming the alkaline phosphatase activity of the SSCs, 10 \u0026micro;L of cell suspension was used to quantify the cell concentration using a hemocytometer. The viability of cells was assessed in triplicate using 30 \u0026micro;L of cell suspension and 5 \u0026micro;L of 0.4% trypan blue (Gibco #15250061, Grand Island, USA). Cell counting was performed using the method suggested by Louis and Siegel (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe cell suspension from the Percoll density gradient centrifugation was used for the differential plating. The culture media was composed of Dulbecco's modified Eagle\u0026rsquo;s medium (high glucose DMEM) (Gibco # 11965, Grand Island, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine (Sigma #G7513, St. Louis, USA), 1 mM pyruvate (Sigma #S8636, St. Louis, USA), 1\u0026times; MEM vitamin solution (Sigma #M6895 St. Louis, USA), 1\u0026times; MEM nonessential amino acid solution (Sigma #M7147, St. Louis, USA), and 1\u0026times; antibiotic and antimycotic solution (Sigma #A5955, St. Louis, USA). The cells were cultured at a concentration of 1.5\u0026times;10\u003csup\u003e7\u003c/sup\u003e SSCs/mL in a 90 x 15 mm sterile Petri dish with agar 1% for 14 h in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and 100% humidity at 30\u0026deg;C (Sanyo Electric, MCO-20AIC, Sakata, Japan). Then, the cells in suspension were recovered, centrifuged at 300 \u0026times; g for 8 minutes at 25\u0026deg;C, and resuspended in supplemented Leibovitz (L-15) medium solution. The percentages of SSCs, viability, and alkaline phosphate activity were determined as described for the cells from the Percoll density gradient centrifugation step. These steps are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of isolated spermatogonial stem cells\u003c/h2\u003e \u003cp\u003eThe testis and cells from the purification steps, a mix of the bands from the 20% and 30% Percoll density gradient centrifugation and differential plating, were sampled. All samples were stored at -80\u0026deg;C until determination of the relative \u003cem\u003eddx4\u003c/em\u003e expression, using \u003cem\u003eβ-actin\u003c/em\u003e as an endogenous gene. \u003cem\u003eDdx4\u003c/em\u003e is considered a marker gene of SSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Primer design for the gene of interest and endogenous gene\u003c/h2\u003e \u003cp\u003ePrimers were designed from the alignment of the \u003cem\u003eβ-actin\u003c/em\u003e and \u003cem\u003eddx4\u003c/em\u003e coding region sequences from different fish species deposited at the National Center for Biotechnology Information (NCBI). \u003cem\u003eAstyanax mexicanus β-actin\u003c/em\u003e and \u003cem\u003eddx4\u003c/em\u003e sequences were input into the Nucleotide BLAST program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and sequences with more significant identities were selected primarily from the Characiforme order and after from others, such as Siluriformes and Cypriniformes. Coding regions were obtained by the Open Reading Frame Finder program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The coding regions were aligned using the Multalign program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://multalin.toulouse.inra.fr/multalin/\u003c/span\u003e\u003cspan address=\"http://multalin.toulouse.inra.fr/multalin/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The conserved regions were preferentially selected for the design of specific primers by the Primer-BLAST program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), using \u003cem\u003eA. mexicanus\u003c/em\u003e sequences as a reference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 cDNA synthesis, primer specificity analysis, and sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA extraction from the samples was performed with TRIzol\u0026reg; Reagent (Ambion #15596026, Carlsbad, USA) following the manufacturer's instructions. RNA was quantified using a QIAxpert spectrophotometer (Qiagen, Hilden, Germany). Before cDNA synthesis, 1 \u0026micro;g of RNA from each sample was treated with DNAse I (Sigma‒Aldrich #SLBR4100 V, St. Louis, USA) and used for first-strand cDNA synthesis with a SuperScript III First-Strand Synthesis System kit (Invitrogen #18080051, Vilnius, Lithuania), according to the manufacturer instructions.\u003c/p\u003e \u003cp\u003eThe cDNA was used as a template for amplifying the regions specified by the \u003cem\u003eβ-actin\u003c/em\u003e and \u003cem\u003eddx4\u003c/em\u003e primers by conventional PCR. PCR was performed by recombinant Taq DNA polymerase (Invitrogen #11615-010, Itapevi, Brazil) according to the manufacturer's instructions using 1 \u0026micro;L of cDNA from the samples and the corresponding designed primers. The cycling parameters used were 95\u0026deg;C for 5 minutes, followed by 35 cycles of amplification (95\u0026deg;C for 45 seconds, 55\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds) with a final step at 72\u0026deg;C for 10 minutes. The reaction was performed in a Multigene thermocycler (Labnet International, Inc.). After amplification, the products were assessed for primer specificities by visualizing them on a 2% agarose gel. The recovered products were separated from the gel using the EZNA Gel Extraction Kit (OMEGA), cloned into the pGEM-T easy vector (Promega), and then sent for sequencing to verify the gene sequence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Relative ddx4 expression analysis\u003c/h2\u003e \u003cp\u003eThe relative \u003cem\u003eddx4\u003c/em\u003e expression analysis was performed using \u003cem\u003eβ-actin\u003c/em\u003e as an endogenous gene. Standard curves for the genes of interest and endogenous genes were established using serial dilution (1:3) of a mixture of cDNA from all samples. Samples were diluted 1:5 for the gene expression analysis. A negative reaction control (NRC) was used for each gene. All samples were evaluated in triplicate; for each reaction, 10 \u0026micro;L of Quanti Nova SYBR Green PCR Master Mix (Qiagen #208054, Hilden, Germany), 8 \u0026micro;L of nuclease-free water, 1 \u0026micro;L of primer mix for target genes, and 1 \u0026micro;L of cDNA were used. The curves were made with each point in duplicate and showed efficiency between 0.9 and 1.1 and R\u0026thinsp;\u0026cong;\u0026thinsp;of 0.99. The primers used in the qPCR are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequences of primers used in qPCR for gene expression of piracanjuba SSCs (\u003cem\u003eBrycon orbygnianus\u003c/em\u003e). Since \u003cem\u003eb-actin\u003c/em\u003e is an endogenous gene and \u003cem\u003eddx4\u003c/em\u003e is specific for spermatogonia.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence (5\u0026acute;-3\u0026acute;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmplicon size (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003e\u0026szlig;-actin\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026acute;- CGTGCTGTCTTCCCATCCA-3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR: 5\u0026acute;- TCACCAACGTAGCTGTCCTTCTG-3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eddx4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026acute;- AAGACCACAGGAACTGAGCG-3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR: 5\u0026acute;- CCCGGTCTCCATGAATGCTT-3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe amplification conditions were 95\u0026deg;C for 2 minutes, followed by 40 cycles of amplification (95\u0026deg;C for 5 seconds and 60\u0026deg;C for 15 seconds). The melting curve was performed from 60 to 95\u0026deg;C. The reaction was carried out in a Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany). Relative gene expression analysis was evaluated by the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method Larionov et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cryopreservation of spermatogonial stem cells\u003c/h2\u003e \u003cp\u003eFor the SSCs isolation and cryopreservation experiments, four adult males approximately four years old, weighing 739.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16 g, were used. Animals were euthanized using a solution of 200 mg/L eugenol. The testes collected had a mean weight of 5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018 g. The purification of SSCs was carried out as described in the \u003cem\u003eprevious section\u003c/em\u003e \u0026ldquo;\u003cem\u003eIsolation of spermatogonial stem cells\u003c/em\u003e\u0026rdquo;. The concentration of SSCs was determined microscopically using a hemocytometer. Before the cryopreservation trials, the cell viability of cultured cells was evaluated using Trypan Blue dye (Gibco #15250061, Grand Island, USA) (see above).\u003c/p\u003e \u003cp\u003eBefore freezing, the cell suspension was diluted 1:1 in cryo-solution using 50 \u0026micro;L of cell suspension at a concentration of 1.4\u0026times;10\u003csup\u003e7\u003c/sup\u003e SSCs/mL and 50 \u0026micro;L of cryoprotectant solution. The cryoprotectant solutions were composed of 100 mM glucose and 0.5% bovine albumin serum (Inlab confidence # 1870, SP, Brazil), and the cryoprotectants ethylene glycol (Sigma # SHBB4592 V, St. Louis, USA), glycerol (Sigma # 72996TMV, St. Louis, USA), dimethylsulfoxide (Sigma # SHBG9653 V, St. Louis, USA), dimethylacetamide (Sigma # STBC4643 V, St. Louis, USA), and propanediol (Sigma # MKBF9063 V, St. Louis, USA). The cryoprotectant concentrations were 2 M, 3 M, and 4 M. The cell suspension and cryoprotectant solutions were diluted 1:1. The mixture of cryo-solutions and cells was loaded into 250-\u0026micro;L cryopreservation straws (IMV Technologies, France) with the total volume adjusted to 70 \u0026micro;L by cutting. The straws were sealed with polyvinyl alcohol (Sigma # 089K0037, St. Louis, USA), placed in cryotubes (Corning Incorporated # 430659, DF, Mexico), and transferred to a freezing container (CoolCell\u0026trade; LX Freezing Container, BioCision). The freezing container containing the straws was transferred to an ultrafreezer at -80\u0026deg;C and kept for two hours (estimated period to reach \u0026minus;\u0026thinsp;80\u0026deg;C). The freezing rate was measured using a thermal recorder (OM-EL-USB-TC, Omega Instruments, Norwalk, USA) with a K-type thermocouple (OM-EL-USB-TC, Omega Instruments\u0026reg;, Norwalk, USA), which was inserted inside the straw. After freezing at -80\u0026deg;C, the cryotubes were immersed in liquid nitrogen at -196\u0026deg;C for storage, approximately for one week.\u003c/p\u003e \u003cp\u003eEach sample was thawed in a water bath at 30\u0026deg;C for one minute. The straw was cut at the ends, and the cell suspension was transferred to a 1.5 mL centrifuge microtube and centrifuged at 350 x g for 5 minutes at 25\u0026deg;C (Hermle Labnet # Z326K, Wehingen, Germany). The supernatant was discarded, the pellet was resuspended in 30 \u0026micro;L of Hank\u0026rsquo;s solution, and 5 \u0026micro;L of Trypan Blue dye was added for subsequent viability analysis, which was performed in triplicates (see above).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Transplantation of spermatogonial stem cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFour eight-year-old male piracanjuba were used as cell donors for the transplantation of SSCs into adult triploid hybrid recipients. The animals were euthanized with 200 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil). The testes were collected and weighed, presenting an average of 0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g. After isolation (see procedures above), SSCs from two donors were used for \u003cem\u003ein vitro\u003c/em\u003e cryopreservation using the best cryoprotectant solution (Propanediol 1M).\u003c/p\u003e \u003cp\u003eThe SSCs were stained using the fluorescent membrane marker PKH26 (Sigma‒Aldrich #MKCK8658, St. Louis, USA). The cell pellet was suspended in 1 mL of supplemented L-15 medium, added to 8 \u0026micro;L of PKH26 dye, and incubated for 10 minutes. After this period, 1 mL of fetal bovine serum was added and incubated for one minute to inactivate the fluorescent marker. The volume of the cell suspension was adjusted to 10 mL with supplemented L-15 medium and centrifuged at 300 \u0026times; g for 5 minutes. Three washes were performed with the same medium to eliminate excess dye and fetal bovine serum.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eSterile male adult triploid hybrid recipients (\u003cem\u003eAstyanax altiparanae\u003c/em\u003e female X \u003cem\u003eAstyanax fasciatus\u003c/em\u003e male) and germ cell-free gonads at 11 months of age were used for the transplantation of SSCs (Piva et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A total of 24 animals were divided into two groups (n\u0026thinsp;=\u0026thinsp;12). In the first group, transplantation was performed using freshly isolated SSCs from the Percoll density gradient centrifugation at a concentration of 2.3\u0026times;10\u003csup\u003e6\u003c/sup\u003e SSCs/mL, resulting in 92% cell viability. In the second group, cryopreserved SSCs at 2.9\u0026times;10\u003csup\u003e6\u003c/sup\u003e SSCs/mL with 88% cell viability from the Percoll density gradient were used in transplantation. An insulin syringe coupled with a tip was used to perform the transplant via the urogenital papilla. Approximately 70 \u0026micro;L of the cell suspension contained approximately 1.9\u0026times;10\u003csup\u003e5\u003c/sup\u003e freshly isolated SSCs, and 2.4\u0026times;10\u003csup\u003e5\u003c/sup\u003e cryopreserved SSCs were injected into each recipient. For transplantation, the recipients were anesthetized in a solution containing 100 mg/L eugenol (Biodynamic #10298550063, Ibipora, Brazil).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Evaluation of chimerism by histochemistry\u003c/h2\u003e \u003cp\u003eThe testes (n\u0026thinsp;=\u0026thinsp;2) of transplanted recipients (freshly isolated SSCs and cryopreserved SSCs) at 10 and 20 days after transplant were collected and fixed in Bouin\u0026rsquo;s fixative (Exodus Cient\u0026iacute;fica #FB08835SO, Sumar\u0026eacute;, Brazil) for 24 hours. The samples were dehydrated using increasing concentrations of ethanol (from 70\u0026ndash;100%) and embedded in paraplast (Sigma # SLBS8607, St. Louis, USA). The paraffin blocks were cut at 5 \u0026micro;m using a microtome equipped with a steel blade (Leica RM2235, Lincolnshire, USA). The cut material was examined by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan) and photographed (NikonDSRi2, Nikon, Tokyo, Japan). The slides were visualized by the fluorescent marker PKH26 and counterstained with hematoxylin and eosin to identify cell types.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were evaluated in triplicate for each cryoprotectant at different molar concentrations. Data were reported as the mean and standard error and analyzed for homogeneity using the Levene test (Brown and Forsythe \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1974\u003c/span\u003e), and for normality, the Cramer‒von Mises test was used (Tamura \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Then, they were evaluated by ANOVA and Tukey\u0026rsquo;s test (α\u0026thinsp;=\u0026thinsp;0.05). Statistical analyses were performed using STATISTICA 7.0 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Histological characterization of the testis of piracanjuba\u003c/h2\u003e \u003cp\u003eThe evaluated testes of the juvenile piracanjuba were small and translucent, presenting a gonadosomatic index (GSI) of 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00. The testes were identified in the development phase. Spermatogonia, a few cysts of spermatocysts, spermatids, and some spermatozoa isolated in the testis were observed in the germinal epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). The testes of adult males collected in the winter were translucent and easily visualized in the abdominal cavity, presenting a GSI of 0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00. A spermatogenesis in the regeneration phase was observed, showing a large number of spermatogonia and some cysts of spermatocysts, spermatids, and isolated spermatozoa in the testis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). The testes of adult males collected in the breeding season were large, firm, and had a blanched color, presenting a GSI of 0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13. The spermatogenesis was active with the spermatozoa in the testicular lumen, where cysts of spermatocytes, spermatids, and a few spermatogonia were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). In addition, the animals released semen when slight manual pressure was applied to the coelomic cavity in the craniocaudal direction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Isolation of spermatogonial stem cells\u003c/h2\u003e \u003cp\u003eThe cell mix from the 20% and 30% bands of the Percoll density gradient centrifugation showed 58.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% SSCs, 30.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% spermatocytes, and 11.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05% sperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). After differential plating, 66.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% SSCs, 33.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% spermatocytes, and 0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% sperm were obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The cell viability of SSCs from the Percoll density gradient centrifugation was 92.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06%, increasing to 94.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13% after plating (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). SSCs from the Percoll density gradient centrifugation and differential plating showed higher alkaline phosphatase activity than spermatocytes when visually compared to the intensity of fluorescence from both cells. This result indicates that the purified cells were SSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Analysis of piracanjuba gene sequences\u003c/h2\u003e \u003cp\u003eThe sequencing of the amplified regions of the \u003cem\u003eBrycon orbignyanus β-actin\u003c/em\u003e and \u003cem\u003eddx4\u003c/em\u003e with 86 and 118 bp showed 99% and 92.37% identity, respectively, for \u003cem\u003eAstyanax mexicanus\u003c/em\u003e sequences (NCBI Reference Sequence: XM_007247821.3; XM_022681259.1). Figures\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B, show the alignments of the \u003cem\u003eddx4\u003c/em\u003e and \u003cem\u003eB-actin\u003c/em\u003e sequences from \u003cem\u003eAstyanax mexicanus\u003c/em\u003e and \u003cem\u003eB. orbignyanus\u003c/em\u003e. The products of the amplifications were visualized on a 2% agarose gel, showing the specificity of the primers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Relative ddx4 expression analysis\u003c/h2\u003e \u003cp\u003eThe standard curves for the \u003cem\u003eβ-actin\u003c/em\u003e and \u003cem\u003eddx4\u003c/em\u003e genes showed amplification efficiencies for the gene of interest and the endogenous control was as expected (0.9 to 1.1). The sampling points were located within the limits of the curves. The melting curve showed only one peak at a specific temperature for each amplification product, where no dimer formation was observed for the evaluated genes. The results of the relative \u003cem\u003eddx4\u003c/em\u003e expression indicated that the expression of \u003cem\u003eddx4\u003c/em\u003e was approximately 3.5 times higher in cells from the Percoll density gradient centrifugation (cell mix from the 20% and 30% bands) than in the testis and cells after differential plating (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This result suggests a decrease in the relative \u003cem\u003eddx4\u003c/em\u003e expression in cultured cells from differential plating, even with higher SSC purity in differential plating.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Viability of spermatogonial stem cells after cryopreservation and freezing rate\u003c/h2\u003e \u003cp\u003eThe freezing rate of each treatment is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The cooling rate was similar between the different cryoprotectant solutions, between a minimum of 6.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 minutes (Etileglycol 1.5M) and a maximum of 9.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72 minutes (DMA 2M) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). While the exothermic peak was observed the solution without cryoprotectant had a lower exothermic peak with \u0026minus;\u0026thinsp;3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 (\u0026deg;C) and a time of 4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 (min) Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As expected, control group cells did not show cell viability after thawing without any cryoprotectant \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;0.0000)\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For 1M propanediol, the cell viability was 94.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85% \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;1.0000)\u003c/em\u003e after thawing (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), this solution was considered the best cryoprotective solution. Followed by Propanediol 1.5M, 1M DMA and 2M DMSO presented a viability of 67.57\u0026thinsp;\u0026plusmn;\u0026thinsp;8.15% \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;0.2842)\u003c/em\u003e, 67.41\u0026thinsp;\u0026plusmn;\u0026thinsp;12.85% \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;0.0001)\u003c/em\u003e and 62.15\u0026thinsp;\u0026plusmn;\u0026thinsp;6.45% \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;1.0000)\u003c/em\u003e respectively after thawing (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Ethylene glycol had viability below 60% in the three molarities with values of 36.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.18% \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;0.9999)\u003c/em\u003e (1M), 35.35\u0026thinsp;\u0026plusmn;\u0026thinsp;13.76% \u003cem\u003e(P\u0026thinsp;=\u0026thinsp;1.0000)\u003c/em\u003e (1.5M), and 45.66\u0026thinsp;\u0026plusmn;\u0026thinsp;8.43% (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.9999\u003c/em\u003e) (2M) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFreezing rates of cryoprotectant solutions used in freezing spermatogonia stem cells of piracanjuba (\u003cem\u003eBrycon orbignyanus\u003c/em\u003e) over two hours at -80\u0026deg;C. The cryoprotectant solutions were loaded in straws and frozen through slow cooling (approximately \u0026minus;\u0026thinsp;1\u0026deg;C/min). Data were obtained from three replicates and are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"16\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eCooling rate (-1\u0026deg;C/min) (20\u0026deg;C to 0\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eFreezing rate (-1\u0026deg;C/min) (0\u0026deg;C to -50\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eFreezing temperature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003eExoterm delay (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e \u003cp\u003eExoterm peak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e4.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-3.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePROPANEDIOL 1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-3.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-11.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePROPANEDIOL 1.5M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-5.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-13.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePROPANEDIOL 2M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-8.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-15.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMA 1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-3.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-10.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMA 1.5M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-6.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-15.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMA 2M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-9.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-18.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMSO 1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-4.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-11.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMSO 1.5M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-5.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-10.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMSO 2M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-8.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-15.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLYCEROL 1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-10.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLYCEROL 1.5M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-6.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-15.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLYCEROL 2M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-6.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-15.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eETILEGLYCOL 1M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-9.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eETILEGLYCOL 1.5M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-11.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eETILEGLYCOL 2M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-8.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e-17.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eViability of SSCs before freezing and post-thawing of piracanjuba (\u003cem\u003eBrycon orbignyanus\u003c/em\u003e). Cell suspensions were diluted 1:1 in each cryo-solution, loaded in straws, and frozen through slow cooling (approximately \u0026minus;\u0026thinsp;1\u0026deg;C/min). Data were obtained from four replicates and are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Values with different letters in the same column indicate a significant difference.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eViability (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003ePRE-FREEZING CONTROL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePROPANEDIOL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.5 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.41\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDMA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.85\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.5 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.47\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.04\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.96\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.5 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.94\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.45\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eGLYCEROL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.07\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.5 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.23\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.5\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eETILENOGLICOL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.18\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.5 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.76\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2 M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.43\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003ePOST-DEFROST CONTROL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026plusmn;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP - value\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0.0161\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Evaluation of SSCs transplantation into the testis of adult triploid hybrid recipients\u003c/h2\u003e \u003cp\u003eIn the testes of recipients transplanted (n\u0026thinsp;=\u0026thinsp;2) with freshly isolated SSCs, PKH26-positive cells were visualized ten days after transplantation (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Through counterstaining with hematoxylin and eosin, the initial development of the germinal epithelium was verified in the testes of the transplanted recipients, given that the triploid hybrids were germ cell-free testes (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). At 20 days after transplantation (n\u0026thinsp;=\u0026thinsp;1) with freshly isolated and cryopreserved SSCs (n\u0026thinsp;=\u0026thinsp;1), the initial spermatogenesis process was verified in the transplanted testes, observing the presence of SSCs and some spermatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-D). Without transplantation, the individuals in the control group had testes without germ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Some of the recipients' animals died in the days following the transplant (n\u0026thinsp;=\u0026thinsp;3) for freshly isolated SSCs and cryopreserved SSCs. After a prolonged period of hormonal inductions, some of the transplanted animals were able to produce sperm, but the molecular analysis was inconclusive due to DNA degradation in the samples. The semen was used for \u003cem\u003ein vitro\u003c/em\u003e fertilization of piracanjuba oocytes, as well as intracytoplasmic microinjection, but embryo development was not observed. In microscopy analysis, a few spermatozoids were found, but they were not activated. Other hormonal inductions were attempted, but they did not result in successful sperm production. The procedures were not included in the material and methods once the results were negative.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe evaluation of spermatogenesis stages in juveniles and adult piracanjuba collected in the winter and summer allowed for the identification of the adequate season and spermatogenesis stage to perform the isolation of SSCs. Adults collected in the winter were allowed to obtain a higher cellular concentration of purified SSCs for later use in cryopreservation or cell transplantation. The testes of juvenile animals were small, presenting a GSI of 0.01 ± 0.00, and even with SSCs, many animals are necessary to obtain an adequate quantity of cells to perform cryopreservation or transplantation in adult animals, as described in other studies (Majhi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Piracanjuba is an endangered species; therefore, the use of juveniles is unsuitable for the isolation of SSCs for cryopreservation or transplantation into adult recipients. In the absence of adults, however, testes from juveniles could be used in tissue cryopreservation and SSC isolation for transplantation in larvae (Marinović et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdult testes collected in June were found in the regeneration stage of spermatogenesis, presenting a GSI of 0.07 ± 0.00. In this stage, SSCs were abundant in the gonads. Therefore, it is the most recommended site to isolate SSCs to reach high quantities of isolated SSCs. In the testes of adults collected in December, spermatozoa were observed in abundance in the lumen, ready to be released, and there was a low quantity of SSCs. Therefore, this stage of spermatogenesis is unfavorable for the isolation of SSCs; once the number of spermatozoa is more significant than SSCs, it makes cellular purification difficult. Studies involving the histological characterization and isolation of SSCs are essential to identify the moment the testes produce higher quantities of SSCs. Identifying this spermatogenesis stage is essential to obtain a higher concentration and better purification of SSCs, which can be used later in cell transplantation, cryopreservation, or \u003cem\u003ein vitro\u003c/em\u003e cultivation (Poursaeid et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zupa et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe regeneration spermatogenesis stage of adult piracanjuba was used to isolate SSCs. SSCs were isolated by enzymatic dissociation, Percoll density gradient centrifugation, and differential plating. This presented a degree of purity after differential plating of approximately 8% greater than the Percoll density gradient centrifugation, also obtaining superior viability. It was possible to eliminate mainly spermatocytes and sperm through differential plating, which was reduced from approximately 11% in the Percoll density gradient centrifugation to 0.78% after differential plating. Differential plating was applied as a purification step for the isolation of SSCs in other species, such as \u003cem\u003eO. mykiss\u003c/em\u003e (Shikina et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003eOreochromis niloticus\u003c/em\u003e (Lacerda et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), \u003cem\u003eRhamdia quelen\u003c/em\u003e (Silva et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and \u003cem\u003eProchilodus lineatus\u003c/em\u003e (Dias et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Differential plating is considered an important step to increase the purity of germ stem cells during the purification step. The purification of SSCs is essential for the generation of high-quality \u003cem\u003ein vitro\u003c/em\u003e genetic banks, which preserve the genetic material of endangered species, such as piracanjuba (\u003cem\u003eB. orbignyanus\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe relative \u003cem\u003eddx4\u003c/em\u003e expression analysis of the testis and cells from the purification steps indicated that the Percoll density gradient centrifugation increased relative gene expression when compared with testis and differential plating. This result may be attributed to the increased concentration of SSCs obtained in the Percoll density gradient centrifugation (20% and 30% bands) compared to the whole testis. A higher percentage of SSCs was obtained in the differential plating step. However, the relative \u003cem\u003eddx4\u003c/em\u003e expression was lower than that of cells from the Percoll density gradient centrifugation. This is because \u003cem\u003eddx4\u003c/em\u003e is expressed in germline cells throughout all stages (Hartung et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). After differential plating, the expression was lower due to the loss of type B spermatogonia and spermatocytes. \u003cem\u003eDdx4\u003c/em\u003e is mainly expressed in SSCs of \u003cem\u003eO. niloticus\u003c/em\u003e, with slight expression observed in primary spermatocytes (Kobayashi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In Neotropical species like \u003cem\u003eR. quelen\u003c/em\u003e and \u003cem\u003eColossoma macropomum\u003c/em\u003e, the \u003cem\u003eddx4\u003c/em\u003e gene is primarily expressed in type A and B spermatogonia, with slight expression in spermatids and no expression in spermatozoa. In \u003cem\u003eC. macropomum, ddx4\u003c/em\u003e expression was reported in type A oogonia and oocytes, probably due to the accumulation of germplasm RNA (Ricci et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vasconcelos et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Borella et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In \u003cem\u003eR. quelen\u003c/em\u003e, other genes showed high expression levels in cell fractions enriched by type A spermatogonia, such as \u003cem\u003eplzf\u003c/em\u003e and \u003cem\u003epou5f3\u003c/em\u003e (Lacerda et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo identify SSCs in Percoll density gradient centrifugation and differential plating, we used alkaline phosphatase observing cells positive for this marker. Previously, alkaline phosphatase has been used to identify type A spermatogonia \u003cem\u003ein vitro\u003c/em\u003e cultures of mice (Van Der Wee et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), as well as in fish such as \u003cem\u003eO. latipes\u003c/em\u003e (Hong et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), \u003cem\u003eEpinephelus coioides\u003c/em\u003e (Zhong et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eOpsariichthys bidens\u003c/em\u003e (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCell cryopreservation is an essential strategy to preserve material with high genetic value. However, freezing is potentially fatal for most living cells, mainly due to the formation of intra- and extracellular ice crystals that lead to cell injury (Chowdhury and Ghosh \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mahmud \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, cells from different tissues or cells within the same tissue may have different rates of water efflux across the cell membrane; thus, the optimal cooling rate needs to be determined for each type of cell to be cryopreserved (Hoon et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The cooling curve is an essential parameter for cell survival, and there is little information about the ideal cooling rate for SSCs (Redden et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this work, different cryoprotectants at different molarities were evaluated, as well as their respective freezing rates for cryopreservation of SSCs isolated from piracanjuba. The best cryoprotectant solution found was 1 M propanediol, presenting a viability of 90.74 ± 0.19 after thawing, a freezing temperature of -3.83 ± 0.14°C, a freezing rate of 28.00 ± 0.4 minutes, and an exothermic peak of -11 ± 1.02°C at 3.18 ± 0.16 minutes.\u003c/p\u003e \u003cp\u003eIsolation and cryopreservation of SSCs have been successfully performed in various fish species. For instance, in \u003cem\u003eR. quelen\u003c/em\u003e, 67% of cell viability was achieved after thawing by using MeOH 1.3 M (Rosa et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, in \u003cem\u003eT. tinca\u003c/em\u003e, the viabilities obtained were 57% using 1.5 M glycerol (Linhartová et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and 50% at 3 M DMSO (Marinović et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In a study by Patra et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), \u003cem\u003eLabeo rohita\u003c/em\u003e had a 70% viability rate after being thawed using 1.4 M DMSO. Meanwhile, Zupa et al. (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that \u003cem\u003eArgyrosomus regius\u003c/em\u003e had a viability rate of 28.5% after being thawed using 10% de DMSO. The cryoprotectant and the molar concentration are species-specific; therefore, cryopreservation protocols are necessary for each species (Pšenička et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The results obtained in this study were highly efficient for the cryopreservation of piracanjuba SSCs, reaching a high percentage of viability compared with the results of other studies. In addition, there are few cryopreservation protocols developed for SSCs of Neotropical fish species; this protocol shows promise for establishing future \u003cem\u003ein vitro\u003c/em\u003e genetic banks.\u003c/p\u003e \u003cp\u003eIn the testes of adult triploid hybrid animals that received transplantation of freshly isolated and cryopreserved SSCs, the cells were visualized up to 20 days after transplantation by histological analysis. The combination of the two sterilization methods, hybridization between species and chromosomal manipulation, was efficient in producing recipient testes without germ cells, making them ideal recipients. Initial development of the germinal epithelium in the testes of the recipients indicated that the transplanted cells were proliferating, mainly due to the presence of spermatogonia and spermatocytes. The fact that cryopreserved germ cells colonize the recipient testes makes it possible to establish procedures that can be used in the reconstitution of piracanjuba in the event of extinction, as was done for other species (Yoshizaki and Lee \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Franěk et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome animals that received transplants were evaluated for semen production by hormonal induction. Despite obtaining sperm, \u003cem\u003ein vitro\u003c/em\u003e fertilization and intracytoplasmic microinjection tests were unsuccessful. Additionally, it was not feasible to determine through molecular analysis whether the semen belonged to piracanjuba. This outcome created uncertainty about whether the sperm cells generated could have originated from the transplanted cells or if numerous hormonal stimulations enabled the receiving gonad to produce endogenous semen. Although it did not successfully obtain functional gamete production from the donor species, this work presents promising results for establishing procedures for the reconstitution of piracanjuba. The procedures performed are the first established for Neotropical species using adult triploid hybrid receptors.\u003c/p\u003e \u003cp\u003eTransplantation of piracanjuba SSCs via urogenital papilla was previously performed using \u003cem\u003eA. altiparanae\u003c/em\u003e male diploid recipients sterilized with busulfan and temperature techniques, producing endogenous and exogenous sperm (de Siqueira-Silva et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In other studies, spermatogenesis was depleted using busulfan treatment, and SSC transplantation was performed, resulting in endogenous and exogenous spermatozoa production (Majhi et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lacerda et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, using 100% sterile animals, as in the case of triploid hybrids in which the testes are constituted only by support cells, is advantageous to these works cited since the competition for space and nutrients between endogenous and exogenous cells is eliminated (Dobrinski \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Therefore, some published works tested the production of recipients completely devoid of germ cells (Piva et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yoshikawa et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ponjarat et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This strategy in some studies was successful in germ stem cell transplantation, where gametes were produced only from the donor species (Takeuchi et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn this study, effective procedures for gene banking were established for the first time in an endangered neotropical fish species, the piracanjuba (\u003cem\u003eB. orbignyanus\u003c/em\u003e). These procedures include isolation, cryopreservation, and transplantation. The procedure was optimized by identifying the period of highest abundance of SSCs in the testis of adult animals. The purification methods which involved Percoll density gradient and differential plating have resulted in high purity and viability of SSCs. It has been observed that the SSCs fractioned have shown high alkaline phosphatase activity in comparison to spermatids and spermatocytes. The relative ddx4 expression analysis has indicated a decrease in cells from differential plating as compared to those from the Percoll density gradient. The best cryoprotectant solution for piracanjuba SSCs was a 1 M propanediol solution. Transplantation of freshly isolated and cryopreserved cells resulted in functional SSCs that developed the germinal epithelium in the testes of adult triploid hybrid recipients. Despite the initial evaluations suggesting low gamete production and no observed fertilization, further investigations are required to achieve functional gamete production. The gene banking procedures developed are innovative and have the potential for application in aquaculture and the conservation of endangered species like piracanjuba.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Authors are grateful to FAPESP (Young Investigator Award #2010/17429-1) which permitted the initial works in the yellowtail tetra and China Three Gorges Brasil (CTG Brasil, Project ANEEL #PD-00387-0418/2019) for the full funding of studies on the species. We also acknowledge CEPTA / ICMBio for kindly providing the facilities and experimental fish.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was supported by the\u0026nbsp;China Three Gorges Brasil (CTG Brasil) (Research \u0026amp; Development,\u0026nbsp;Project ANEEL #PD-00387-0418/2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during the current study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were conducted following the Animal Ethics Committee from the National Center for Research and Conservation of Continental Aquatic Biodiversity (CEUA/CEPTA # 02031.000088/2021-61). The authenticity of the document can be verified on the website https://sei.icmbio.gov.br/autenticidade informing the verification code: \u003cstrong\u003e9643544\u0026nbsp;\u003c/strong\u003eand the code\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eCRC: \u003cstrong\u003eEC53598A\u003c/strong\u003e.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdamov NS de M, Nascimento NF do, Maciel ECS, et al (2017) Triploid Induction in the Yellowtail Tetra, Astyanax altiparanae, Using Temperature Shock: Tools for Conservation and Aquaculture. Journal of the World Aquaculture Society 48:741\u0026ndash;750. https://doi.org/10.1111/jwas.12390\u003c/li\u003e\n\u003cli\u003eAgostinho, \u0026Acirc;. A., Thomaz, S. M., \u0026amp; Gomes LC (2005) Conserva\u0026ccedil;\u0026atilde;o da biodiversidade em \u0026aacute;guas continentais do Brasil. Megadiversidade 1:70\u0026ndash;78\u003c/li\u003e\n\u003cli\u003eBORBA MR, FRACALOSSI DM, PEZZATO LE (2006) Dietary energy requirement of piracanjuba fingerlings, Brycon orbignyanus, and relative utilization of dietary carbohydrate and lipid. Aquaculture Nutrition 12:183\u0026ndash;191. https://doi.org/10.1111/j.1365-2095.2006.00401.x\u003c/li\u003e\n\u003cli\u003eBorella MI, Chehade C, Costa FG, et al (2020) The brain-pituitary-gonad axis and the gametogenesis. In: Biology and Physiology of Freshwater Neotropical Fish. Elsevier, pp 315\u0026ndash;341\u003c/li\u003e\n\u003cli\u003eBrinster CJ, Ryu BY, Avarbock MR, et al (2003) Restoration of fertility by germ cell transplantation requires effective recipient preparation. Biology of Reproduction 69:412\u0026ndash;420. https://doi.org/10.1095/biolreprod.103.016519\u003c/li\u003e\n\u003cli\u003eBrown-Peterson NJ, Wyanski DM, Saborido-Rey F, et al (2011) A standardized terminology for describing reproductive development in fishes. Marine and Coastal Fisheries 3:52\u0026ndash;70. https://doi.org/10.1080/19425120.2011.555724\u003c/li\u003e\n\u003cli\u003eBrown MB, Forsythe AB (1974) Robust tests for the equality of variances. Journal of the American Statistical Association 69:364\u0026ndash;367. https://doi.org/10.1080/01621459.1974.10482955\u003c/li\u003e\n\u003cli\u003eChen X, Kan Y, Zhong Y, et al (2022) Generation of a Normal Long-Term-Cultured Chinese Hook Snout Carp Spermatogonial Stem Cell Line Capable of Sperm Production In Vitro. Biology 11:. https://doi.org/10.3390/biology11071069\u003c/li\u003e\n\u003cli\u003eChowdhury S, Ghosh S (2021) Cryopreservation of Stem Cells. In: Stem Cells. Springer Singapore, Singapore, pp 81\u0026ndash;105\u003c/li\u003e\n\u003cli\u003eCinalli RM, Rangan P, Lehmann R (2008) Germ Cells Are Forever. Cell 132:559\u0026ndash;562. https://doi.org/10.1016/j.cell.2008.02.003\u003c/li\u003e\n\u003cli\u003ede Siqueira-Silva DH, dos Santos Silva AP, da Silva Costa R, et al (2019) Preliminary study on testicular germ cell isolation and transplantation in an endangered endemic species Brycon orbignyanus (Characiformes: Characidae). Fish Physiology and Biochemistry. https://doi.org/10.1007/s10695-019-00631-8\u003c/li\u003e\n\u003cli\u003eDias GCM, Cassel M, Jesus LWO De, Borella MI (2020) Isolation , in vitro study , and stem cell markers for type A spermatogonia in a Characiformes species. 1\u0026ndash;17. https://doi.org/10.1002/mrd.23394\u003c/li\u003e\n\u003cli\u003eDo Nascimento NF, Bertolini RM, Lopez LS, et al (2021) Heat-induced triploids in Brycon amazonicus: A strategic fish species for aquaculture and conservation. Zygote. https://doi.org/10.1017/S0967199421000125\u003c/li\u003e\n\u003cli\u003eDobrinski I (2006) Transplantation of germ line stem cells for the study and manipulation of spermatogenesis. Ernst Schering Research Foundation workshop 175\u0026ndash;193. https://doi.org/10.1007/3-540-31437-7_12\u003c/li\u003e\n\u003cli\u003eFraněk R, Cheng Y, Fuč\u0026iacute;kov\u0026aacute; M, et al (2022) Who is the best surrogate for germ stem cell transplantation in fish? Aquaculture 549:. https://doi.org/10.1016/j.aquaculture.2021.737759\u003c/li\u003e\n\u003cli\u003eFraněk R, Marinović Z, Lujić J, et al (2019) Cryopreservation and transplantation of common carp spermatogonia. PLoS ONE 14:1\u0026ndash;17. https://doi.org/10.1371/journal.pone.0205481\u003c/li\u003e\n\u003cli\u003eFujihara R, Katayama N, Sadaie S, et al (2022) Production of Germ Cell-Less Rainbow Trout by dead end Gene Knockout and their Use as Recipients for Germ Cell Transplantation. Marine Biotechnology 417\u0026ndash;429. https://doi.org/10.1007/s10126-022-10128-w\u003c/li\u003e\n\u003cli\u003eHartung O, Forbes MM, Marlow FL (2014) Zebrafish vasa is required for germ-cell differentiation and maintenance. Molecular Reproduction and Development 81:946\u0026ndash;961. https://doi.org/10.1002/mrd.22414\u003c/li\u003e\n\u003cli\u003eHong Y, Liu T, Zhao H, et al (2004) Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proceedings of the National Academy of Sciences of the United States of America 101:8011\u0026ndash;8016. https://doi.org/10.1073/pnas.0308668101\u003c/li\u003e\n\u003cli\u003eHoon T, Park SC, Yang JH, et al (2017) Cryopreservation and its clinical applications. Integrative Medicine Research 6:12\u0026ndash;18. https://doi.org/10.1016/j.imr.2016.12.001\u003c/li\u003e\n\u003cli\u003eICMBio (2018) Livro Vermelho da Fauna Brasileira Amea\u0026ccedil;ada de Extin\u0026ccedil;\u0026atilde;o. Bras\u0026iacute;lia: ICMBio.\u003c/li\u003e\n\u003cli\u003eKobayashi T, Kajiura-Kobayashi H, Nagahama Y (2000) Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus. Mechanisms of Development 99:139\u0026ndash;142. https://doi.org/10.1016/S0925-4773(00)00464-0\u003c/li\u003e\n\u003cli\u003eLabb\u0026eacute; C, Robles V, Herraez MP (2013) Cryopreservation of gametes for aquaculture and alternative cell sources for genome preservation. Advances in Aquaculture Hatchery Technology 76\u0026ndash;116. https://doi.org/10.1533/9780857097460.1.76\u003c/li\u003e\n\u003cli\u003eLacerda, S.R. Batlouni, S.B.G. Silva, C.S.P. Homem LRF (2006) Germ cell type Days. In: Anim Reprod. p 2\u003c/li\u003e\n\u003cli\u003eLacerda SM dos SN, Costa GMJ, de Fran\u0026ccedil;a LR (2014) Biology and identity of fish spermatogonial stem cell. General and Comparative Endocrinology 207:56\u0026ndash;65. https://doi.org/10.1016/j.ygcen.2014.06.018\u003c/li\u003e\n\u003cli\u003eLacerda SMSN, Batlouni SR, Assis LH, et al (2008) Germ cell transplantation in tilapia (Oreochromis niloticus). Cybium 32:115\u0026ndash;118\u003c/li\u003e\n\u003cli\u003eLacerda SMSN, Batlouni SR, Costa GMJ, et al (2010) A new and fast technique to generate offspring after germ cells transplantation in adult fish: The nile tilapia (Oreochromis niloticus) model. PLoS ONE 5:1\u0026ndash;9. https://doi.org/10.1371/journal.pone.0010740\u003c/li\u003e\n\u003cli\u003eLacerda SMSN, Martinez ERM, Mura ILDD, et al (2019) Duration of spermatogenesis and identification of spermatogonial stem cell markers in a Neotropical catfish, Jundi\u0026aacute; (Rhamdia quelen). General and Comparative Endocrinology 273:249\u0026ndash;259. https://doi.org/10.1016/j.ygcen.2018.10.018\u003c/li\u003e\n\u003cli\u003eLarionov A, Krause A, Miller WR (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinformatics 6:1\u0026ndash;16. https://doi.org/10.1186/1471-2105-6-62\u003c/li\u003e\n\u003cli\u003eLinhartov\u0026aacute; Z, Rodina M, Guralp H, et al (2014) Isolation and cryopreservation of early stages of germ cells of tench (Tinca tinca). Czech Journal of Animal Science 59:381\u0026ndash;390\u003c/li\u003e\n\u003cli\u003eLopera-barrero NM (2007) Diversidade Genetica de Brycon orbignyanus. Universidade Estadual de Maring\u0026aacute; Centro de Ci\u0026ecirc;ncias Agr\u0026aacute;rias\u003c/li\u003e\n\u003cli\u003eLouis KS, Siegel AC (2011) Cell Viability Analysis Using Trypan Blue: Manual and Automated Methods. In: Stoddart MJ (ed) Mammalian cell viability: methods and protocols. Humana Press, Totowa, NJ, pp 7\u0026ndash;12\u003c/li\u003e\n\u003cli\u003eMahmud N (2021) Cryopreservation of Stem Cell Product. In: Chandy M, Radhakrishnan VS, Sukumaran RK (eds). Springer International Publishing, Cham, pp 499\u0026ndash;507\u003c/li\u003e\n\u003cli\u003eMajhi SK, Hattori RS, Rahman SM, Str\u0026uuml;ssmann CA (2014) Surrogate production of eggs and sperm by intrapapillary transplantation of germ cells in cytoablated adult fish. PLoS ONE 9:. https://doi.org/10.1371/journal.pone.0095294\u003c/li\u003e\n\u003cli\u003eMajhi SK, Hattori RS, Yokota M, et al (2009) Germ cell transplantation using sexually competent fish: An approach for rapid propagation of endangered and valuable germlines. PLoS ONE 4:1\u0026ndash;8. https://doi.org/10.1371/journal.pone.0006132\u003c/li\u003e\n\u003cli\u003eMajhi SK, Rasal AR, Kushwaha B, Raizada S (2017) Heat and chemical treatments in adult Cyprinus carpio (Pisces cypriniformes) rapidly produce sterile gonads. Animal Reproduction Science 183:77\u0026ndash;85. https://doi.org/10.1016/j.anireprosci.2017.05.015\u003c/li\u003e\n\u003cli\u003eMarinović Z, Li Q, Lujić J, et al (2019) Preservation of zebrafish genetic resources through testis cryopreservation and spermatogonia transplantation. Scientific Reports 9:1\u0026ndash;10. https://doi.org/10.1038/s41598-019-50169-1\u003c/li\u003e\n\u003cli\u003eMarinović Z, Lujić J, K\u0026aacute;sa E, et al (2017) Cryosurvival of isolated testicular cells and testicular tissue of tench Tinca tinca and goldfish Carassius auratus following slow-rate freezing. General and Comparative Endocrinology 245:77\u0026ndash;83. https://doi.org/10.1016/j.ygcen.2016.07.005\u003c/li\u003e\n\u003cli\u003eNozu R, Nakamura M (2020) Influence of prolonged cultivation on sexual characteristics of sterilized female tilapia, Oreochromis mossambicus, induced by high-temperature treatment. Aquaculture 524:735245. https://doi.org/10.1016/j.aquaculture.2020.735245\u003c/li\u003e\n\u003cli\u003eOkutsu T, Suzuki K, Takeuchi Y, et al (2006) Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish. Proceedings of the National Academy of Sciences of the United States of America 103:2725\u0026ndash;2729. https://doi.org/10.1073/pnas.0509218103\u003c/li\u003e\n\u003cli\u003ePandit NP, Bhandari RK, Kobayashi Y, Nakamura M (2015) High temperature-induced sterility in the female Nile tilapia, Oreochromis niloticus. General and Comparative Endocrinology 213:110\u0026ndash;117. https://doi.org/10.1016/j.ygcen.2015.01.028\u003c/li\u003e\n\u003cli\u003ePatra S, Mishra G, Dash SK, et al (2016) Transplantation worthiness of cryopreserved germ cells of Indian major carp rohu, Labeo rohita. Current Science 111:739\u0026ndash;746. https://doi.org/10.18520/cs/v111/i4/739-746\u003c/li\u003e\n\u003cli\u003ePiva LH, de Siqueira-Silva DH, Goes CAG, et al (2018) Triploid or hybrid tetra: Which is the ideal sterile host for surrogate technology? Theriogenology 108:239\u0026ndash;244. https://doi.org/10.1016/j.theriogenology.2017.12.013\u003c/li\u003e\n\u003cli\u003ePonjarat J, Singchat W, Monkheang P, et al (2019) Evidence of dramatic sterility in F 1 male hybrid catfish [male Clarias gariepinus (Burchell, 1822) \u0026times; female C. macrocephalus (G\u0026uuml;nther, 1864)] resulting from the failure of homologous chromosome pairing in meiosis I. Aquaculture 505:84\u0026ndash;91. https://doi.org/10.1016/j.aquaculture.2019.02.035\u003c/li\u003e\n\u003cli\u003ePoursaeid S, Kalbassi MR, Hassani SN, Baharvand H (2020) Isolation, characterization, in vitro expansion and transplantation of Caspian trout (Salmo caspius) type a spermatogonia. General and Comparative Endocrinology 289:113341. https://doi.org/10.1016/j.ygcen.2019.113341\u003c/li\u003e\n\u003cli\u003eP\u0026scaron;enička M, Saito T, Rodina M, Dzyuba B (2016) Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells. Cryobiology 72:119\u0026ndash;122. https://doi.org/10.1016/j.cryobiol.2016.02.005\u003c/li\u003e\n\u003cli\u003eRedden E, Davey R, Borjigin U, et al (2009) Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia. Cryobiology 58:190\u0026ndash;195. https://doi.org/10.1016/j.cryobiol.2008.12.005\u003c/li\u003e\n\u003cli\u003eRicci JMB, Martinez ERM, Butzge AJ, et al (2018) Characterization of vasa homolog in a neotropical catfish, Jundi\u0026aacute; (Rhamdia quelen): Molecular cloning and expression analysis during embryonic and larval development. Gene 654:116\u0026ndash;126. https://doi.org/10.1016/j.gene.2018.02.029\u003c/li\u003e\n\u003cli\u003eRobles V, Cabrita E, Paz Herr\u0026aacute;ez M (2009) Germplasm cryobanking in zebrafish and other aquarium model species. Zebrafish 6:281\u0026ndash;293. https://doi.org/10.1089/zeb.2009.0592\u003c/li\u003e\n\u003cli\u003eRodriguez-Rodriguez M del P, Lopera-Barrero NM, Ribeiro RP, et al (2010) Diversidad gen\u0026eacute;tica de piracanjuba usada en programas de repoblaci\u0026oacute;n con marcadores microsat\u0026eacute;lites. Pesquisa Agropecu\u0026aacute;ria Brasileira 45:56\u0026ndash;63. https://doi.org/10.1590/s0100-204x2010000100008\u003c/li\u003e\n\u003cli\u003eRosa IF, Martinez ERM, Digmayer M, et al (2023) Successful Cryopreservation of Spermatogonia Stem Cells of Neotropical Catfish (Rhamdia quelen) and Enriched Germ Cell Transplantation into Common Carp (Cyprinus carpio) Testes. Fishes 8:1\u0026ndash;13. https://doi.org/10.3390/fishes8100478\u003c/li\u003e\n\u003cli\u003eSeki S, Kusano K, Lee S, et al (2017) Production of the medaka derived from vitrified whole testes by germ cell transplantation. Scientific Reports 7:1\u0026ndash;11. https://doi.org/10.1038/srep43185\u003c/li\u003e\n\u003cli\u003eShikina S, Ihara S, Yoshizaki G (2008) Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia. Molecular Reproduction and Development 75:529\u0026ndash;537. https://doi.org/10.1002/mrd.20771\u003c/li\u003e\n\u003cli\u003eSilva MA, Costa GMJ, Lacerda SMSN, et al (2016) Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes. General and Comparative Endocrinology 230\u0026ndash;231:48\u0026ndash;56. https://doi.org/10.1016/j.ygcen.2016.03.012\u003c/li\u003e\n\u003cli\u003eTakeuchi Y, Yatabe T, Yoshikawa H, et al (2018) Production of functionally sterile triploid Nibe croaker Nibea mitsukurii induced by cold-shock treatment with special emphasis on triploid aptitude as surrogate broodstock. Aquaculture 494:45\u0026ndash;56. https://doi.org/10.1016/j.aquaculture.2016.05.030\u003c/li\u003e\n\u003cli\u003eTakeuchi Y, Yoshizaki G, Takeuchi T (2001) Production of germ-line chimeras in rainbow trout by blastomere transplantation. Molecular Reproduction and Development 59:380\u0026ndash;389. https://doi.org/10.1002/mrd.1044\u003c/li\u003e\n\u003cli\u003eTakeuchi Y, Yoshizaki G, Takeuchi T (2003) Generation of Live Fry from Intraperitoneally Transplanted Primordial Germ Cells in Rainbow Trout 1. 1149:1142\u0026ndash;1149. https://doi.org/10.1095/biolreprod.103.017624\u003c/li\u003e\n\u003cli\u003eTamura H (2008) 済無No Title No Title. Journal of Chemical Information and Modeling 53:287. https://doi.org/10.1017/CBO9781107415324.004\u003c/li\u003e\n\u003cli\u003eVan Der Wee KS, Johnson EW, Dirami G, et al (2001) Immunomagnetic isolation and long-term culture of mouse type a spermatogonia. Journal of Andrology 22:696\u0026ndash;704. https://doi.org/10.1002/j.1939-4640.2001.tb02230.x\u003c/li\u003e\n\u003cli\u003eVasconcelos ACN, Streit DP, Octavera A, et al (2019) Isolation and characterization of a germ cell marker in teleost fish Colossoma macropomum. Gene 683:54\u0026ndash;60. https://doi.org/10.1016/j.gene.2018.10.027\u003c/li\u003e\n\u003cli\u003eViveiros ATM, Orf\u0026atilde;o LH, Nascimento AF, et al (2012) Effects of extenders, cryoprotectants and freezing methods on sperm quality of the threatened Brazilian freshwater fish pirapitinga-do-sul Brycon opalinus (Characiformes). Theriogenology 78:361\u0026ndash;368. https://doi.org/10.1016/j.theriogenology.2012.02.015\u003c/li\u003e\n\u003cli\u003eXu D, Yoshino T, De Bello Cioffi M, et al (2020) Production of donor-derived eggs after ovarian germ cell transplantation into the gonads of adult, germ cell-less, triploid hybrid fish. Biology of Reproduction 103:1289\u0026ndash;1299. https://doi.org/10.1093/biolre/ioaa168\u003c/li\u003e\n\u003cli\u003eXu D, Yoshino T, Konishi J, et al (2019) Germ cell-less hybrid fish: Ideal recipient for spermatogonial transplantation for the rapid production of donor-derived sperm. Biology of Reproduction 101:492\u0026ndash;500. https://doi.org/10.1093/biolre/ioz045\u003c/li\u003e\n\u003cli\u003eYasui GS, Fujimoto T, Arai K (2010) Restoration of the loach, Misgurnus anguillicaudatus, from cryopreserved diploid sperm and induced androgenesis. Aquaculture 308:S140\u0026ndash;S144. https://doi.org/10.1016/j.aquaculture.2010.05.041\u003c/li\u003e\n\u003cli\u003eYoshikawa H, Ino Y, Shigenaga K, et al (2018) Production of tiger puffer Takifugu rubripes from cryopreserved testicular germ cells using surrogate broodstock technology. Aquaculture 493:302\u0026ndash;313. https://doi.org/10.1016/j.aquaculture.2018.05.016\u003c/li\u003e\n\u003cli\u003eYoshizaki G, Lee S (2018) Production of live fish derived from frozen germ cells via germ cell transplantation. Stem Cell Research 29:103\u0026ndash;110. https://doi.org/10.1016/j.scr.2018.03.015\u003c/li\u003e\n\u003cli\u003eZaniboni Filho E, Reynalte-Tataje D, Weingartner M (2006) Potencialidad del g\u0026eacute;nero Brycon en la piscicultura brasile\u0026ntilde;a. Revista Colombiana de Ciencias Pecuarias 19:233\u0026ndash;240\u003c/li\u003e\n\u003cli\u003eZardo \u0026Eacute;L, Fornari DC, Gioria J, et al (2021) Gonadal development period and sexual differentiation through histological analysis in Brycon orbignyanus (Valenciennes, 1850) (Characiformes: Bryconidae). Aquaculture 539:. https://doi.org/10.1016/j.aquaculture.2021.736636\u003c/li\u003e\n\u003cli\u003eZhong C, Tao Y, Liu M, et al (2022) Establishment of a Spermatogonial Stem Cell Line with Potential of Meiosis in a Hermaphroditic Fish, Epinephelus coioides. Cells 11:. https://doi.org/10.3390/cells11182868\u003c/li\u003e\n\u003cli\u003eZhou L, Wang X, Liu Q, et al (2021) Successful Spermatogonial Stem Cells Transplantation within Pleuronectiformes: First Breakthrough at inter-family Level in Marine Fish. International Journal of Biological Sciences 17:4426\u0026ndash;4441. https://doi.org/10.7150/ijbs.63266\u003c/li\u003e\n\u003cli\u003eZupa R, Martino NA, Marzano G, et al (2020) Meagre argyrosomus regius (Asso, 1801) stem spermatogonia: Histological characterization, immunostaining, in vitro proliferation, and cryopreservation. Animals 10:. https://doi.org/10.3390/ani10050851\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"conservation, spermatogonia, transplantation, gene bank, germline chimera","lastPublishedDoi":"10.21203/rs.3.rs-4266695/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4266695/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePiracanjuba (\u003cem\u003eBrycon orbignyanus\u003c/em\u003e) is an endangered fish species from the Neotropical region. The establishment of a cryobank using spermatogonial stem cells (SSCs) and subsequent production of a germline chimera is thus a promising strategy for such species. In the present work, procedures for the isolation and cryopreservation of piracanjuba SSCs and subsequent transplantation into sterile recipients were established. The piracanjuba SSCs were obtained by Percoll density gradient centrifugation and differential plating. SSC fractions were evaluated by relative \u003cem\u003eddx4\u003c/em\u003e expression, alkaline phosphatase activity, and light microscopy. SSC cryopreservation was performed using five cryoprotectants at three different concentrations. The mix of the cells from the 20% and 30% Percoll density gradients showed 58.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% purity of SSCs. The purity of SSCs increased to 66.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01% after differential plating. The relative \u003cem\u003eddx4\u003c/em\u003e expression was 3.5 times higher in cells from the Percoll density gradient centrifugation than in the gonad and cells after differential plating. Propanediol (1M) was the most effective cryoprotector evaluated (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;1.000\u003c/em\u003e), showing 90.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85% cell viability. Freshly isolated and cryopreserved cells from the Percoll density gradient centrifugation were transplanted into a sterile male adult triploid hybrid with germ cell-less gonads. SSCs were observed in the germinal epithelium of the testes of recipients 20 days after transplantation. The results are promising for obtaining functional germline chimeras in Neotropical fish. Consequently, the procedures established here can be applied in future actions for the conservation and reconstitution of the piracanjuba in case of extinction.\u003c/p\u003e","manuscriptTitle":"Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 16:28:47","doi":"10.21203/rs.3.rs-4266695/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T06:21:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T12:20:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"072799f4-4fdc-4f33-8a7b-1612f67cf662","date":"2024-04-28T05:32:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"bdbe0f6a-3963-4e4f-ad19-4c4ecfd20032","date":"2024-04-23T11:40:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-23T04:55:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-20T18:45:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-17T11:59:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2024-04-15T02:00:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ffa1e1dd-1e16-44e4-ad02-4043d8727687","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:10:43+00:00","versionOfRecord":{"articleIdentity":"rs-4266695","link":"https://doi.org/10.1007/s10695-024-01406-6","journal":{"identity":"fish-physiology-and-biochemistry","isVorOnly":false,"title":"Fish Physiology and Biochemistry"},"publishedOn":"2024-09-27 15:57:44","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-04-25 16:28:47","video":"","vorDoi":"10.1007/s10695-024-01406-6","vorDoiUrl":"https://doi.org/10.1007/s10695-024-01406-6","workflowStages":[]},"version":"v1","identity":"rs-4266695","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4266695","identity":"rs-4266695","version":["v1"]},"buildId":"veTbxFhMMB0_faC6-Wkog","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}