Introduction
Fish spermatogenesis is a highly regulated biological process that leads to the production of functional spermatozoa (Schulz and Nóbrega, Reference Schulz, Nóbrega and Farrell2012; de Siqueira-Silva et al., Reference de Siqueira-Silva, da Silva Rodrigues and Nóbrega2019a). Type A undifferentiated spermatogonia(SSCs) play an essential role in maintaining active spermatogenesis, thereby ensuring male fertility, due to their capacity for self-renewal, differentiation, and transfer of genetic information (NÓBREGA et al., Reference Nóbrega, Greebe, van de Kant, Bogerd, de França and Schulz2010; Angel A A Vigoya et al., Reference Vigoya, Martinez, Digmayer, Oliveira, Butzge, Rosa, Doretto and Rafael2024). Therefore, their unique biological properties have positioned them as ideal candidates for innovative approaches to species recovery. One such promising biotechnological strategy is the transplantation of germline cells, which has shown potential for species reconstitution (Lacerda et al., Reference Lacerda, Costa, Campos-Junior, Segatelli, Yazawa, Takeuchi, Morita, Yoshizaki and França2013; de Siqueira-Silva et al., Reference de Siqueira-Silva, Saito, dos Santos-Silva, da Silva Costa, Psenicka and Yasui2018). This procedure involves transferring germline cells to sterile recipients to optimize reproduction through surrogate propagation (Okutsu et al., Reference Okutsu, Shikina, Kanno and Takeuchi2007; Pšenička et al., Reference Pšenička, Saito, Linhartová and Gazo2015). This strategy has been applied to species such as Nile Tilapia (Oreochromis niloticus) (Lacerda et al., Reference Lacerda, Batlouni, Costa, Segatelli, Quirino, Queiroz, Kalapothakis and França2010), pejerrey (Odontesthes bonariensis) (Majhi et al., Reference Majhi, Hattori, Rahman and Strüssmann2014), blue drum (Nibea mitsukurii) (Xu et al., Reference Xu, Yoshino, De Bello Cioffi, Yoshikawa, Ino, Yazawa, Lacerda and Takeuchi2020), and goldfish (Carassius auratus) (Majhi, Reference Majhi2023). Given its conservation potential, germline transplantation represents a valuable tool for species of ecological and environmental concern.
The piracanjuba (Brycon orbignyanus), the donor species used in the present study, exemplifies the need for such biotechnological approaches. This migratory fish, native to the Uruguay and Paraná river basins (Zaniboni-Filho and Schulz, Reference Zaniboni-Filho, Schulz, Carolsfeld, Harvey, Ross and Baer2003), is currently listed as endangered (EN- A2c as reported by ICMBio, (2022) due to anthropic activities, such as riparian deforestation, dam constructions, pollution, and the introduction of alien species (ICMBio, 2018). Piracanjuba is a key species for the maintenance of riparian forests because it acts as a seed disperser. Additionally, the species is sensitive to environmental conditions, serving as a bio-indicator of environmental conditions (Lopera-barrero, Reference Lopera-barrero2007). Therefore, previous research efforts on artificial reproduction were conducted to maintain both of natural and ex-situ resources. However, limitations such as delayed sexual maturation and the rise of predominance of male offspring have hindered the success of re-stocking programs (Lopera-barrero, Reference Lopera-barrero2007; Rodriguez-Rodriguez et al., Reference Rodriguez-Rodriguez, Lopera-Barrero, Ribeiro, Povh, Vargas, Sirol and Jacometo2010; Zardo et al., Reference Zardo, Fornari, Gioria, Rotili, Gomes, Esquivel-Muelbert and Streit2021). These limitations reinforce the need for alternative strategies such as surrogate propagation to support the conservation and recovery of this and other threatened Neotropical species.
To achieve successful surrogate propagation, ensuring complete sterility of the recipient species is crucial, as this guarantees that only heterologous gametes will be produced by the resultant germline chimera. Several strategies for host sterilization have been implemented, including techniques such as triploidization (Adamov et al., Reference Adamov, Nascimento, Maciel, Pereira-Santos, Senhorini, Calado, Evangelista, Nakaghi, Guerrero, Fujimoto and Yasui2017; Bertolini et al., Reference Bertolini, Lopez, do Nascimento, Arashiro, de Siqueira Silva, dos Santos, Senhorini and Yasui2020; Yasui et al., Reference Yasui, Nakaghi, Monzani, Nascimento, Pereira dos Santos, Goes, Porto-Foresti and Senhorini2020), hybridization between species (Xu et al., Reference Xu, Yoshino, Konishi, Yoshikawa, Ino, Yazawa, Dos Santos Nassif Lacerda, De França and Takeuchi2019; Hu et al., Reference Hu, Chu, Xiong, Mei, Lin, Ren, Huang and Guo2021), UV-irradiation (Saito et al., Reference Saito, Güralp, Iegorova, Rodina and Pšeniaka2018), and chemical sterilization using busulfan at high temperatures (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, Ninhaus-Silveira and Veríssimo-Silveira2015; Majhi et al., Reference Majhi, Maurya, Kumar, Mohindra and Lal2019), as well as the combination of techniques, such as hybridization and triploidization (Piva et al., Reference Piva, de Siqueira-Silva, Goes, Fujimoto, Saito, Dragone, Senhorini, Porto-Foresti, Ferraz and Yasui2018; Xu et al., Reference Xu, Yoshino, De Bello Cioffi, Yoshikawa, Ino, Yazawa, Lacerda and Takeuchi2020).
In our previous studies (Yasui et al., Reference Yasui, Ferreira do Nascimento, Pereira-Santos, Santos Silva, Coelho, Visintin, Porto-Foresti, Okada Nakaghi, Vianna, Carvalho, Monzani, López and Senhorini2022), the yellowtail tetra (Astyanax altiparanae) was used as a host model organism for surrogate technology by producing triploid animals (Adamov et al., Reference Adamov, Nascimento, Maciel, Pereira-Santos, Senhorini, Calado, Evangelista, Nakaghi, Guerrero, Fujimoto and Yasui2017). However, 100% sterile offspring, with no gametogenesis, were confirmed when hybrid triploids (Piva et al., Reference Piva, de Siqueira-Silva, Goes, Fujimoto, Saito, Dragone, Senhorini, Porto-Foresti, Ferraz and Yasui2018) were produced. Histological observations of the sterile triploids indicate that germ cell lineage is still present in the gonads, demonstrating the ability for proliferation, and maintenance. However, they lost the ability to undergo meiosis and proceed with gametogenesis (Ferreira Do Nascimento et al., Reference Ferreira Do Nascimento, De Siqueira-Silva, Pereira-Santos, Fujimoto, Senhorini, Nakaghi and Yasui2017). For surrogate technology, the presence of endogenous germ cells in the host may decrease the efficacy of germ cell transplantation. This is because the presence of these cells hinders the entry of exogenous cells transplanted through the urogenital papilla in adult animals, and the transplanted cells may compete for colonization of the niches and subsequent maintenance by the supporting Sertoli and Leyd cells. Thus, transplanted cells compete not only for space but also for the supporting cells crucial for maintenance during gametogenesis. Therefore, depleting endogenous germ cells, even in sterile triploid fish, may be important to enhance the success of surrogate propagation.
Given this, sterilization strategies such as chemical treatment with busulfan associated with high water temperatures represent a promising method to empty the gonads, as previously performed in diploid animals (Lacerda et al., Reference Lacerda, Batlouni, Costa, Segatelli, Quirino, Queiroz, Kalapothakis and França2010; NóBREGA et al., Reference Nóbrega, Greebe, van de Kant, Bogerd, de França and Schulz2010; de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, Ninhaus-Silveira and Veríssimo-Silveira2015), thereby facilitating the entry of transplanted cells. Based on this approach, the objective of this study was to obtain a completely sterile yellowtail tetra (Astyanax altiparanae) and to transplant the stem SSCs of B. orbignyanus. Specifically, this study aimed to produce diplod and tripoid of yellowtail tetra (A. altiparanae) as recipients for piracanjuba (B. orbignyanus) germline cells, to identify the most effective sterilization method and the optimal recipient type, and ultimately to generate germline chimeras.
To achieve these objectives, three separate experiments were conducted, as shown in the supplementary material. Experiments 1 and 2 were focused on inducing sterility in diploid and triploid males of A. altiparanae using the chemical treatment of busulfan associated with high water temperature (35°C). In experiment 3, stem SSCs from B. orbignyanus were purified and transplanted into recipients whose busulfan treatment was effective (longer sterility time). The transplantation was evaluated using histological and fluorescence microscopy techniques.
Materials and methods
Animal collection and ethics statement
Fish sampling and experiments were conducted 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 following the Animal Ethics Committee from CEPTA (CEUA/CEPTA #02031.000088/2021-61).
Experiment 1: depletion of spermatogenesis in diploid of Astyanax altiparanae
Animal and acclimatization protocols
Sexually mature diploid males A. altiparanae (n = 192), were used for complete sterilization using a protocol combining high water temperature and the chemical agent busulfan. Animals with an average body weight of 7.10 ± 0.39 g and 65.53 ± 1.25 mm standard length were used. Fish were maintained in 60-L aquaria (16 fish per aquaria; three replicates per treatment) under a closed recirculation system with aeration, controlled temperature, and photoperiod (12L: 12D), and fed twice daily.
Fish were acclimated at 28°C for one week, after which temperature was increased by 1°C per day until it reached 35°C and maintained for one week prior to chemical treatment (Figure 1A). Ten days after the final busulfan application, the temperature was gradually reduced to 28°C, were fish were maintained for an additional month.
Effect of treatment with busulfan associated with high temperature on the gonadosomatic index in diploid adult males of Astyanax altiparanae. In statistical analyses, no significant differences were observed between treatments. Data were described as mean ± standard error.
Figure 1 Long description
The bar graph titled Busulfan treatment in diploid males compares the gonadosomatic index percentage across different collection days for four treatment groups. The x-axis represents collection days at intervals of 28, 38, 58, 78, and 108 days. The y-axis represents the gonadosomatic index percentage, ranging from 0 to 4 percentage. There are four data series represented by different colors: blue for C 15, gray for C 40, dark blue for B 15, and light blue for B 40. Each bar represents the mean gonadosomatic index percentage for each treatment group at the specified collection days. The graph shows variations in the gonadosomatic index across different treatment groups and collection days. All values are approximated.
Experimental procedures
The procedures were carried out in accordance with previously published works of Majhi et al., Reference Majhi, Rasal, Kushwaha and Raizada(2017) and de Siqueira-Silva et al., (Reference de Siqueira-Silva, dos Santos Silva, Ninhaus-Silveira and Veríssimo-Silveira2015). The busulfan (Sigma Aldrich, St. Louis, USA) was first dissolved in dimetilsulfóxido (DMSO) (Sigma #472301, St. Louis, USA) at 37°C, and then diluted in Ringer’s solution (128.3 mM NaCl, 23.6 mM KCl, 3.6 mM CaCl2, 2.1 mM MgCl2) to prepare working concentrations [15 mg kg−1 (B15) and 40 mg kg−1 (B40)]. The experiment was divided into four treatments: the control received only the vehicle DMSO using the same concentrations of busulfan [15 mg kg−1 (C15) and 40 mg kg−1 (C40)], and two treatments with busulfan B15 and B40.
After the acclimation period at 35°C, the fish received five applications of DMSO and chemical treatment busulfan with an interval of 10 days between each application. Busulfan injections were administered through the intraperitoneal cavity, using an insulin syringe. Before applying the injections, the fish were anesthetized using an eugenol solution (100 mg L−1).
Confirmation of spermatogenesis depletion
Before starting the experiments, testes of A. altiparanae (n = 2) from each treatment were collected at 28°C to confirm that the animals were sexually mature. Aiming to confirm sterility, testes from two animals per treatment were collected on the 28th, 38th, 58th, 78th, and 108th days after the last treatment injection. All fish were euthanized in a eugenol solution (200 mg L−1). Their biometric data were recorded and the gonadosomatic index (GSI) was calculated using the equation GSI = GW/BW × 100, where GW is the gonad weight, and BW is the total body weight of the fish. Testes were collected and fixed in Bouin’s solution for 24 h and then stored in 70% ethanol for histological processing. The samples were initially dehydrated in increasing concentrations of ethanol (70, 80, 90, and 100%), then clarified in xylene solution (Scientific Exodus #X09738RA, Sumaré, Brazil), and finally included in paraplast (Sigma #SLBS8607, St. Louis, USA). The blocks obtained were cut into 5-μm thick sections using a rotative manual microtome (Leica Biosystems #RM2235, Heidelberger, Germany). The slides were stained with Hematoxylin and eosin, analyzed by optical microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and photographed using a digital camera (Nikon DS-Ri2, Nikon, Tokyo, Japan), mounted on the microscope. Digital images were captured using Nis-Ar Elements (Nikon, Tokyo, Japan) software.
Experiment 2: depletion of spermatogenesis in triploids juvenile of Astyanax altiparanae
Broodstock induction and triploidization
The gametes were collected from A. altiparanae broodstock, and in vitro fertilization was performed as previously reported by Yasui et al. (Reference Yasui, Senhorini, Shimoda, Pereira-Santos, Nakaghi, Fujimoto, Arias-Rodriguez and Silva2015). Briefly, females and males were injected with carp pituitary. Females received two doses: the first dose of 0.3 mg kg−1 and the second of 3 mg kg−1 six h afterward. In males, only one dose of 3 mg kg−1 was performed, at the time of the second induction dose for females. Six h after the second hormonal induction, the gametes were collected separately, mixed with water at 26°C, and then underwent triploidization according to the protocol of Adamov et al., Reference Adamov, Nascimento, Maciel, Pereira-Santos, Senhorini, Calado, Evangelista, Nakaghi, Guerrero, Fujimoto and Yasui2017. The fertilized eggs of A. altiparanae were heat-shocked at 40°C two min after fertilization for two min, returned to the water at 26°C, and placed in sieves inside aquariums of 60-L with constant aeration. Three days after hatching, the larvae were fed with newly hatched brine shrimp nauplii, as previously reported by Bertolini et al. (Reference Bertolini, Senhorini, Nascimento, Pereira-Santos, Nakaghi, Peres, Silva and Yasui2018). After the larvae started eating powdered feed, they were transferred to 8 × 8 m2 fishponds and fed twice daily.
Triploidization analysis
Two months after hatching, the animals (n = 120) were removed from the fishponds and allocated to 60-L aquariums (n = 10) at 28°C with constant aeration. The animals were then subjected to flow cytometry analyses to confirm ploidy, following the protocol described by Xavier et al. (Reference Xavier, Senhorini, Pereira-Santos, Fujimoto, Shimoda, Silva, dos Santos and Yasui2017). All fish were anesthetized in an eugenol solution (100 mg L−1). A small part of the caudal fin of each animal was cut and transferred individually to a 1.5-mL microtube containing 120-μL of detergent solution (9.53 mM MgSO4.7 H2O; 47.67 mM KCl; 15 mM Tris; pH 8.0, 74 mM sucrose, and Triton 0,6%) for 10 min. Then, the nuclei were stained, and 800-μL of a solution of 4,6 Dimidine 2 Phenylidone Di-Hydrochloride – DAPI 0.01% (DAPI em Dulbecco’s Phosphate Buffer Saline) was added. The samples were filtered through 30-μm nylon screens and analyzed in a flow cytometer (CyFlow Ploidy Analyzer, Partec, GMBh, and Alemanha) using A. altiparanae semen as a reference sample. Triploid animals were selected and used in Experimental procedures in juvenile triploid of A. altiparanae.
Experimental procedures in juvenile triploid of A. altiparanae
In experiment 2, ninety-six two-month-old triploid juvenile males of A. altiparanae, with an average body mass of 7.83 ± 0.26 g and 70.28 ± 1.06 mm standard length, were used for complete sterilization using the chemical agent busulfan. The experiments were carried out based on experiment 1 and results obtained in other studies with the species (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, Ninhaus-Silveira and Veríssimo-Silveira2015; Postingel Quirino et al., Reference Postingel Quirino, da Silva Rodrigues, da Silva Cabral, de Siqueira-Silva, Mori, Butzge, Nóbrega, Ninhaus-Silveira and Veríssimo-Silveira2021). Two experimental groups were used: a control group (n = 24) that was kept at 35°C and a group that was sterilized with busulfan (n = 72). The preparation of the busulfan solution, and the application method, was carried out according to the protocol previously described in Experiment 2: depletion of spermatogenesis in triploids juvenile of Astyanax altiparanae . Three administrations of busulfan (50 mg kg−1) were performed for each fish with seven-day intervals between each application. Seven days after the last busulfan application, the temperature was gradually reduced to 28°C, where fish were maintained for an additional month.
Confirmation of spermatogenesis depletion
After seven days of acclimatization at 28°C, the testes of two males were collected for the identification of spermatogenesis. One week after the fish were acclimated to 35°C, the testes of two males were collected and used to identify the spermatogenesis stage before beginning the sterilization procedures. The testes of two animals per treatment were collected after the second and third busulfan application and one month after finishing the busulfan treatment. The biometric data from each animal were measured, including the weight, standard length, total length, and gonad weight. Sample collection, fixation, processing, and analysis were carried out following the procedure described previously in section Broodstock induction and Triploidization.
Statistical analysis
GSI data from each experiment (1 and 2) were reported as mean and standard error. The data were analyzed for homogeneity, using the test of Levene, (Brown and Forsythe, Reference Brown and Forsythe1974), and for normality, the Cramer-von Mises test was used (‘Summary for Policymakers’, 2014). They were then evaluated by ANOVA and Tukey’s test (α = 0.05). Statistical analyses were performed using STATISTICA 7.0 software.
Experiment 3: transplantation of SSCs stem cells in triploid recipients of A. altiparanae previously treated with busulfan
Enzymatic dissociation and isolation of stem SSCs
Two male B. orbignyanus adults, at 8 years old, weighing 0.93 ± 0.07 kg and with a standard length of 38.25 ± 0.88 cm, were used for SSCs isolation. SSCs isolation was carried out in June, a period when the testes of B. orbignyanus are in the regeneration phase (Figure 7A). The procedures for isolation, purification, and identification were carried out following the methodology previously described by Siqueira et al. (Reference de Siqueira-Silva, dos Santos Silva, da Silva Costa, Senhorini, Ninhaus-Silveira and Veríssimo-Silveira2019b) and López et al. (Reference López, Monzani, Carvalho, de Siqueira Silva, Vianna, Yasui and Senhorini2024). The collected testes were processed individually. For the enzymatic dissociation process, the collected testes were transferred to a 90 × 15 mm sterile Petri dish containing Leibovitz (L-15) cell culture medium (Gibco #21083027, Grand Island, USA) supplemented with 0.5% fetal bovine serum (Gibco #12657029, Grand Island, USA), 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). The testes were minced using scalpel blades and washed three times with supplemented Leibovitz (L-15) medium to remove excess blood cells.
The minced testes were 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−1 collagenase type I from Clostridium histolyticum (Sigma #C0130, St. Louis, USA). Enzymatic dissociation was performed for 2 h at room temperature in a rotary shaker (Agitador Phenix, São Paulo, Brazil). Afterward, 20-µg mL DNase I (Sigma‒Aldrich #SLBT5559, St. Louis, USA) was added to the solution and incubated for another hour under the same conditions. The cell suspension was filtered through a 50-µm pore diameter nylon mesh to eliminate undegraded tissues and transferred to a 15-mL centrifuge tube. The final volume was adjusted to 10-mL with supplemented Leibovitz (L-15) medium and centrifuged at 300 × g for 5 min (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 min to remove collagenase and DNase I. This step was performed twice.
SSCs stem cells were fractionated from spermatids, sperm, and somatic cells by Percoll density gradient at 10, 20, 30, and 40% Percoll® Plus (Sigma‒Aldrich #SLBH8181 V, St. Louis, USA). The Percoll density gradient was made by layering 1 mL of each Percoll solution in a 15-mL centrifuge tube from the highest to the lowest concentration of Percoll. The cell suspension was carefully deposited on the surface of the 10% density gradient Percoll solution and centrifuged at 800 × g for 30 min at 25°C. The collected bands were resuspended in 10- mL of supplemented Leibovitz (L-15) medium and centrifuged at 300 × g for 8 min at 25°C to remove the Percoll. The cells deposited at the bottom of the centrifuge tubes were suspended in 1 mL of supplemented Leibovitz (L-15) medium. Cell suspensions were microscopically analyzed to verify which fractions contained the highest percentage of SSCs.
SSCs were visualized in the 20 and 30% bands of the Percoll density gradients. By labeling with alkaline phosphatase it was possible to identify the SSCs, the labeling procedures were carried out following the manufacturer’s protocol (Life Technologies #A14353, Frederick, USA). Fluorescent cells were verified by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and digital images were obtained (Nikon DS-Ri2, Nikon, Tokyo, Japan). To evaluate the SSCs concentration, the bands containing SSCs were mixed (20 and 30%). Evaluation of the concentration and purity of SSCs was performed in a Neubauer chamber. The SSCs were verified in triplicate using 30-µL of cell suspension mixed with 5-µL of 0.4% trypan blue (Gibco #15250061, Grand Island, USA) following the protocol of Louis and Siegel (Reference Louis, Siegel and Stoddart2011).
PKH26 labeling of spermatogonia stem cells
The SSCs were stained using the fluorescent membrane labeling PKH26 (Sigma‒Aldrich #MKCK8658, St. Louis, USA). The cell pellet was suspended in 1 mL of supplemented L-15 medium, 8-µL of PKH26 dye was added, incubated for 10 min at room temperature, 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 min. This wash was performed three times to remove excess dye and fetal bovine serum.
Transplantation
The SSCs transplantation was performed in 27 triploid males of A. altiparanae at 3.5 months of age, previously treated with busulfan. PKH26-Labeled SSCs were used for transplantation at 2.6 × 106 SSCs/mL, with 93% cell viability. Approximately 70-μL of the cell suspension containing approximately 1.82 × 105 SSCs was picked up using an insulin syringe attached to a tip and injected into each recipient via the urogenital papilla (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, da Silva Costa, Senhorini, Ninhaus-Silveira and Veríssimo-Silveira2019b). Before transplantation, the recipients were anesthetized in eugenol solution at 100 mg L−1.
Evaluation of chimerism by histochemistry
The testes of the recipient animals were collected at 23, 42 days (n = 4), and 80 days after transplantation (n = 3). The collected samples were fixed in Bouin (Exodus Científica #FB08835SO, Sumaré, Brazil) for 24 h and later transferred to 70% alcohol. The samples were subjected to dehydration using increasing concentrations of ethanol (70, 80, 90, and 100%) and then embedded in paraplast. The blocks were cut at a thickness of 5-μm using a microtome equipped with a steel blade. The histological slides were evaluated by fluorescence microscopy (Nikon-Eclipse Ni, Tokyo, Japan), and the slides were counterstained with hematoxylin and eosin for identification of PKH26-positive cells. PKH26-positive fields were photographed using a CCD camera (DS-Ri2, Nikon, Tokyo, Japan). Digital images were captured using Nis-Ar Elements (Nikon, Tokyo, Japan).
Results
Experiment 1: depletion of spermatogenesis in diploid of A. altiparanae
GSI analysis and external morphological characteristics of the testes
Statistical analyses in GSI assessments between treatments C15, C40, B15, and B40 showed no significant differences (Figure 1). The external morphological characteristics of the testes of groups C15 and C40 became visually more translucent after starting the experiment (28th) (Figure 2A, B) and ten days after applying the last application of busulfan (78th) (Figure 2E, F). After the temperature decrease period to 28°C (108th), the testes of the control groups returned to a whitish color, suggesting the return of spermatogenesis (Figure 2I, J). In the treatment B15 and B40, the testes collected at 28th days of experiments, presented the same external morphologies as the control groups, well developed and with a whitish color (Figure 2C, D). On the 78th day of the experiment, after the fifth application of busulfan, the treatments B15 and B40 presented translucent and regressed testes (Figure 2G,H) concerning the beginning of treatment with busulfan. A more pronounced regression was visually observed in treatment B40 (Figure 2H) in relation to B15 treatment (Figure 2G). At 108th days of the experiment, 30 days after finishing treatment with busulfan and kept at 28°C, the testes of individuals in treatment B15 and B40 no longer presented the appearance of regressed gonads, as in Figure 3G, H, observing translucent and yellow parts in the testes (Figure 2K, L).
Representative photographs of Astyanax altiparane testes, throughout busulfan treatment on different collection days. Characteristics such as aspects of the color, reduction and growth of the testes were visualized. The testes from the four treatments presented a translucent appearance during the acclimatization period at 35°C (A, B, C, D). After five busulfan applications (78th), a reduction in testes was observed in treatments with busulfan B15 (G) and B40 (H), when compared with the control groups C15 (E) and C40 (F). One month after finishing the experiment (108th), in groups treated with busulfan (K–L), the appearance of the testes is similar to control groups (I–J), indicating the return of spermatogenesis. Scale bars: 0.5 cm.
Figure 2 Long description
Twelve photos of Astyanax altiparane testes during busulfan treatment on different collection days. The testes from the four treatments presented a translucent appearance during the acclimatization period at 35 degrees Celsius. After five busulfan applications, a reduction in testes was observed in treatments with busulfan B15 and B40, when compared with the control groups C15 and C40. One month after finishing the experiment, in groups treated with busulfan, the appearance of the testes is similar to control groups, indicating the return of spermatogenesis.
Testes of diploid Astyanax altiparanae, from the control groups (C15 and C40) and groups treated with busulfan (B15 and B40), at 28th, 38th and 58th days of the experiment. Testes collected before performing the first busulfan application (A–D), kept for a week at 35°C. Testes collected after the first busulfan application (E–H). Testes collected after the third busulfan application (I–L). Sg: Spermatogonia; Sp, Spermatocytes; St, Spermatids; Sz, Sperm; L, Testicular lumen; Arrowhead, Pyknotic cell nucleus; Asterisk, Sperm being reabsorbed. Scale bars: A–D; I–J: 100 μm; E–H; K–L: 50 μm.
Figure 3 Long description
The image displays testes of diploid Astyanax altiparanae from control groups and groups treated with busulfan at different days of the experiment. The testes are shown at the 28th, 38th, and 58th days. Various stages of spermatogenesis are labeled, including spermatogonia, spermatocytes, spermatids, and sperm. The testicular lumen and pyknotic cell nuclei are also indicated. The image highlights the differences in testicular structure and cell types across different treatment groups and time points.
Effects of busulfan and high temperature on the spermatogenesis
The testes of the fish collected on day 28th of the experiment presented active spermatogenesis. The presence of SSCs, spermatocytes, spermatids, and spermatozoa in the testicular lumen was observed in the germinal epithelium (Figure 3A–D). On day 38th of the experiment, in the control C15, and C40, SSCs, spermatocytes, spermatids in the germinal epithelium, and spermatozoa being reabsorbed in the testicular lumen were visualized (Figure 3E, F). In treatment B15, some SSCs, spermatocytes, and cells with pyknotic nuclei were observed in the germinal epithelium (Figure 3G). In treatment B40, some spermatocytes, and cells with pyknotic nuclei were observed in the germinal epithelium, as well as a degeneration of the germinal epithelium and an increase in the area of testicular lumen (Figure 3H).
On day 58th of the experiment, the control C15, and C40 had SSCs, spermatocytes, and spermatids in the germinal epithelium. In some areas of the testes, sperm reabsorption was observed in the testicular lumen (Figure 3I, J). In the B15 treatment, the suppression of spermatogenesis was almost complete, with some SSCs and spermatocytes being observed in the germinal epithelium and large areas of the testicular lumen free of spermatozoa (Figure 3K). In treatment B40, only the presence of testicular lumen and interstitium was observed in the testes (Figure 3L), indicating complete depletion of spermatogenesis.
On day 78th of the experiment, after the fifth application of busulfan, the control C15 already showed the return of the spermatogenesis process, germline cells were observed in the germinal epithelium and spermatozoa in the testicular lumen (Figure 4A). In the control C40, spermatocytes and spermatids were observed in the testes (Figure 4B). In the B15 treatment, some spermatocytes and spermatids were visualized (Figure 4C). In treatment B40, the depletion of spermatogenesis was complete, and only testicular lumen being observed (Figure 4D).
Testes of diploid Astyanax altiparanae, from the control groups (C15 and C40) and groups treated with busulfan (B15 and B40), at 78th and 108nd days of the experiment. Testes collected after the fifth busulfan application (A–D). Testes collected one month after the end of treatment with busulfan (E–L). Sg, Spermatogonia; Sp, Spermatocytes; St, Spermatids; Sz, Sperm; L, Testicular lumen; Arrowhead, Pyknotic cell nucleus and Asterisk, Sperm being reabsorbed. Scale bars: A, B: 100 μm; C–D; E–L: 50 μm.
Figure 4 Long description
The image displays microscopic sections of fish testes from diploid Astyanax altiparanae, divided into control groups (C15 and C40) and groups treated with busulfan (B15 and B40). The sections are observed at the 78th and 108th days of the experiment. Key stages of spermatogenesis are labeled, including spermatogonia (Sg), spermatocytes (Sp), spermatids (St), and sperm (Sz). The testicular lumen (L) is also visible. Specific features such as pyknotic cell nuclei (indicated by an arrowhead) and sperm being reabsorbed (indicated by an asterisk) are highlighted. The scale bars for images A and B are 100 micrometers, while for images C, D, E, F, G, H, I, J, K, and L, the scale bars are 50 micrometers.
On day 108th of the experiment, one month after the end of busulfan treatment, the testes of the C15 and C40 control groups showed active spermatogenesis, presenting all germline cells (SSCs, spermatocytes, and spermatids in the germinal epithelium and spermatozoa in the testicular lumen) (Figure 4E, F). In treatment B15, the presence of some isolated SSCs was noted (Figure 4I, J), in some individuals in the cranial part of the testes, only a few SSCs and spermatids were observed in the germinal epithelium and a few spermatozoa in the testicular lumen (Figure 4G), suggesting the return of spermatogenesis. In treatment B40, some SSCs and spermatids were observed in the germinal epithelium and spermatozoa in the testicular lumen (Figure 4H). Some individuals still had complete spermatogenesis depletion (Figure 4K, L), suggesting that the higher concentration of busulfan used retards the return of spermatogenesis.
Experiment 2: depletion of spermatogenesis in juvenile triploid of A. altiparanae
The testes collected on day 28th of the experiment (before the first application of busulfan) showed a yellow-translucent color and a GSI of 2.39 ± 0.18 (Figure 5A, B, I). At 42nd days of the experiment, the testes collected from the control group had a translucent appearance in some parts (Figure 5C) with a GSI of 2.50 ± 0.19 (Figure 5I). In the busulfan-treated group, the testes were all translucent (Figure 5D), showing a reduction in size and GSI 1.11 ± 0.37 (Figure 5I). At 49th days of the experiment, the testes of the individuals collected in the control group showed a whitish color, which was more pronounced than those of the control group at the 42nd day of the experiment (Figure 5E), with 3.17 ± 0.97 GSI (Figure 5I). In this collection, the testes of the fish treated with busulfan, were completely translucent (Figure 5F), and the GSI had the lowest calculated value among all the evaluated samples (0.79 ± 0.04 (Figure 5I).
Effect of busulfan treatment at high temperature (35°C) on the morphological characteristics of the testes, and the gonadosomatic index in triploid juvenile of Astyanax altiparanae from day 28th (before the first application of busulfan) and the control and busulfan treatment groups at 42th, 49th and 85th days of the experiment. Testes during acclimation at 28°C (A). Before the first application of busulfan (35°C) (B). Testes of the control group at 42th days of the experiment (C). Testes of the busulfan-treated group at 42th days of the experiment (D). Testes of the control group on the 49th day of the experiment (E). Testes of the busulfan-treated group at 49th days of the experiment (F). Testes of the control group on the 85th day of the experiment (G). Testes of the busulfan-treated group on the 85th day of the experiment (H). I: Effect of busulfan treatment at high temperature (35°C) on the gonadosomatic index in triploid juvenile of A. altiparanae male. Data are presented as the mean ± standard error. Scale bars: 0.5 cm.
Figure 5 Long description
The image contains eight photos of testes and one bar graph. The photos show testes at different stages of an experiment involving busulfan treatment at high temperature. The stages include acclimation at 28 degrees Celsius, before the first application of busulfan at 35 degrees Celsius, and various days of the experiment for both control and busulfan-treated groups. The bar graph illustrates the effect of busulfan treatment on the gonadosomatic index in triploid juvenile Astyanax altiparanae males over different collection days. The data are presented as mean standard error.
At 85th day of the experiment, 30 days after the last application of busulfan, in the testes of the control group, parts with a yellowish color were observed (Figure 5G), and an increase in testes size to 4.13 ± 0.27 of GSI was observed (Figure 5I). In the group treated with busulfan, the testes presented a translucent aspect in the anterior part, while the posterior region had a whitish color (Figure 5H), and an increase in GSI (1.82 ± 0.21) was observed. A GSI increase was observed for both groups; however, in the group treated with busulfan, a lower value was noted (Figure 5I).
The testes collected from the control group on the 7th and 28th days of the experiment corresponded to the acclimation stage at 28 and 35°C. Germinal epithelium cysts of SSCs, spermatocytes, and Sertoli cells were observed (Figure 6A, B). On the 42nd day of the experiment, in the testes of the control group, spermatocytes were observed in some parts, cells with a pyknotic nucleus, and the testicular lumen was devoid of sperm (Figure 6C). In the group treated with busulfan, the testes showed complete depletion of germ cells, with interstitial cells and testicular lumen free of sperm (Figure 6D, E).
Testes of triploids juvenile of Astyanax altiparanae, before and during treatment with busulfan. Testes of control at 7 days of the experiment during acclimation at 28°C (A). Testes of the control group at 28 days of the experiment and after acclimatization to 35°C (B). On the 42th day of the experiment, the testes of control group (C). Testes treated with busulfan (D). Higher magnification image for identification of Sertoli cells (E). On the 49th day of the experiment, after the third application of busulfan. Testes of control group (F). Testes treated with busulfan (G). Higher magnification image for identification of Sertoli cells (H). On the 85th day of the experiment, one month after the end of the treatment with busulfan. Testes of control group (I). Testes treated with busulfan (J–K). Sg, spermatogonia; Sp, spermatocytes; St, spermatids; Cp, cell with a pyknotic nucleus; L, testicular lumen; I, interstice tissue; White arrow with black outline, Sertoli cell. Scale bars: A–B: 100 μm; contoured area: 50 μm; C, F, G: 200 μm; D–I, J, K: 100 μm; E, H: 50 μm.
Figure 6 Long description
The image presents a detailed cross-sectional view of testes from triploid juvenile Astyanax altiparanae, illustrating the effects of busulfan treatment over time. The testes are shown at different stages: before treatment, during acclimation at 28 degrees Celsius, after acclimatization to 35 degrees Celsius, and at various days post-treatment. Key labeled parts include spermatogonia (Sg), spermatocytes (Sp), spermatids (St), cells with pyknotic nuclei (Cp), testicular lumen (L), and interstice tissue (I). Higher magnification images highlight the identification of Sertoli cells. The scale bars indicate measurements in micrometers, providing context for the size of the observed structures.
At 49th days of the experiment, the testes of the control group still had spermatocytes and cells with a pyknotic nucleus. However, the testicular lumen was devoid of sperm (Figure 6F). In the busulfan-treated group, the collected testes were devoid of germ cells and sperm, and only testicular lumen and interstitium were observed in the histological slides of the testes (Figure 6G, H). One month after the end of the treatment with busulfan, at 85th day of the experiment, the presence of SSCs, spermatocytes, and spermatids in the germinal epithelium was observed in the testes of individuals in the control group. However, the presence of sperm was not observed in the testicular lumen (Figure 6I). In the busulfan-treated group, half of the individuals analyzed (n = 2) presented testes free of germ cells and sperm, and only the testicular lumen and interstitial cells were observed (Figure 6J). In the other half of the individuals, the testes (n = 2) presented some SSCs stem cells along the germinal epithelium (Figure 6K).
Spermatogonial stem cell transplantation in triploid animals treated with busulfan
The B. orbignyanus SSCs were previously identified using alkaline phosphatase (Figure 7D, E). SSCs transplantation was performed in 27 triploid juveniles of A. altiparanae treated with busulfan (Figure 7F). However, 10 recipients died some days after transplantation. On the day of transplantation, the non-transplanted control testes were free of germ cells, presenting only testicular lumen, interstitium, and Sertoli cells (Figure 7B, C). At 23 days after transplantation, PKH26-positive cells were observed in the testes of recipients (n = 3) (Figures 7G and 8B). The staining with hematoxylin and eosin evidenced SSCs cysts distributed by all germinal epithelium (Figures 7H–J and 8B). At 42 days after transplantation, PKH26-labeled cells were also visualized in the recipients’ testes (n = 3) (Figure 8C). Hematoxylin and eosin staining of the slides indicated PKH26-positive cells as SSCs stem cells and some spermatocytes (Figure 8D). At 80 days after transplantation all the three recipients collected, presented SSCs stem cells and spermatocytes labeled with distributed throughout the germinal epithelium (Figure 8E, F).
Testes of adult Brycon orbignyanus collected in June, a period when a greater number of spermatogonia can be found (A). After cell isolation by Percoll gradient, spermatogonia were identified using alkaline phosphatase. Visualization by optical microscopy (D) and fluorescence microscopy (E). Testes visualized at lower magnification, confirming complete depletion of spermatogenesis by treatment with busulfan (B). Higher magnification view of the region of figure (B), identifying the presence of Sertoli cells (C). Transplantation of B. orbignyanus spermatogonial stem cells via urogenital papilla in triploid juvenile of Astyanax altiparanae previously treated with busulfan (F). White dotted lines indicate regions containing PKH26-labeled spermatogonia visualized by fluorescence microscopy (G). Light microscopy analysis of the regions delimited in G, with the slide stained with hematoxylin and eosin (H–J). Scale bars: B: 400 μm; A, G, H: 200 μm; C, D, E–I, J: 50 μm.
Figure 7 Long description
The collage consists of ten images. The first image (A) shows testes of adult Brycon orbignyanus collected in June, highlighting spermatogonia. The second image (B) shows testes at lower magnification, confirming complete depletion of spermatogenesis by treatment with busulfan. The third image (C) is a higher magnification view of the region in B, identifying the presence of Sertoli cells. The fourth image (D) shows spermatogonia identified using alkaline phosphatase and visualized by optical microscopy. The fifth image (E) shows spermatogonia visualized by fluorescence microscopy. The sixth image (F) depicts the transplantation of B. orbignyanus spermatogonial stem cells via urogenital papilla in triploid juvenile of Astyanax altiparanae previously treated with busulfan. The seventh image (G) shows regions containing PKH26-labeled spermatogonia visualized by fluorescence microscopy. The eighth, ninth, and tenth images (H, I, J) show light microscopy analysis of the regions delimited in G, with the slide stained with hematoxylin and eosin.
Testes of triploids Astyanax altiparanae analyzed by fluorescence and optical microscopy at 23 (A–B), 42 (C–D) and 80 (E–F) days after transplantation. White dotted lines indicate regions containing PKH26-positive germ cells visualized by fluorescence microscopy (A, C, E). Light microscopy analysis of the regions delimited in A, C and E with the slide stained with hematoxylin and eosin, showing the germ cells (B, D and F). Scale bars: B: 100 μm; A, C–F: 50 μm.
Figure 8 Long description
The image consists of six panels labeled A to F, showing testes of triploid Astyanax altiparanae analyzed by fluorescence and optical microscopy at 23, 42, and 80 days after transplantation. Panels A, C, and E display regions containing PKH26-positive germ cells visualized by fluorescence microscopy, with white dotted lines indicating these regions. Panels B, D, and F show light microscopy analysis of the regions delimited in A, C, and E, respectively, with slides stained with hematoxylin and eosin, highlighting the germ cells. The scale bars indicate measurements of 100 micrometers for panel B and 50 micrometers for panels A, C, and F.
Discussion
One of the objectives of this work was to achieve complete sterilization in males A. altiparanae. In experiment 1, five administrations of busulfan at dosages (15 mg kg−1 and 40 mg kg−1) under high temperature conditions (35°C) effectively depleted spermatogenesis in adult diploid males. However, spermatogenesis resumed one month after treatment, indicating that the sterilization effect was temporary. Recovery occurred more rapidly in the 15 mg kg−1 group, where spermatozoa were already present in the testicular lumen, whereas in the 40 mg kg−1 group only SSCs and some spermatid cysts were observed. Previous studies on A. altiparanae reported that two busulfan doses (18 mg kg−1 and 15 mg kg−1) administered 14 days apart resulted in complete depletion of spermatogenesis. However, spermatogenesis was rapidly reactivated, with endogenous spermatozoa later observed in the testicular lumen after transplantation of B. orbignyanus SSCs (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, Ninhaus-Silveira and Veríssimo-Silveira2015, Reference de Siqueira-Silva, dos Santos Silva, da Silva Costa, Senhorini, Ninhaus-Silveira and Veríssimo-Silveira2019b). These data corroborate the results obtained in the study using a dosage of 15 mg kg−1.
In other published work on the carp (Cyprinus carpio) (Majhi et al., Reference Majhi, Rasal, Kushwaha and Raizada2017) and catfish (Pangasianodon hypophthalmus) (Majhi et al., Reference Majhi, Maurya, Kumar, Mohindra and Lal2019), using a dosage of 40 mg kg−1 of busulfan in five applications, complete depletion of spermatogenesis was obtained for a long period (about 4 months after the end of busulfan treatment). In this study, the dose of 40 mg kg−1 of busulfan in adult males of A. altiparanae only delayed the return of spermatogenesis compared to the lower dose. The presence of SSCs one month after the end of the experiment indicates the return of spermatogenesis. It is worth mentioning that A. altiparanae is precocious for reproduction (capable of reproduction in 4 months) and spawns several times annually (Porto-Foresti et al., Reference Porto-Foresti, Castilho-Almeida, Senhorini and Foresti2010). These distinct aspects of A. altiparanae compared to carp and catfish may contribute to the differences in the return of spermatogenesis observed in these studies.
Based on the results from adult diploid of A. Altiparanae (experiment 1), a strategy was developed for immature diploid juveniles, which are in the initial phase of spermatogenesis, to prevent or delay the early return of spermatogenesis. Therefore, the busulfan concentration was increased, and the application interval was reduced to 7 days to determine the optimal dosage for undifferentiated diploid juveniles of A. altiparanae. For this, doses of 40 mg kg−1, 50 mg kg−1, and 60 mg kg−1 of busulfan were used. The results were inconclusive, as the fish died after the third application of busulfan, with only a few fish surviving from the 50 mg kg−1 concentration. Nevertheless, at a dosage of 50 mg kg−1 of busulfan after two applications, complete depletion of spermatogenesis was observed. In dosages of 40 mg kg−1 and 60 mg kg−1 of busulfan, only ovaries were collected, preventing a comparison of dosages in males treated with 50 mg kg−1 (not published data). Other species, such as the young olive flounder (Paralichthys olivaceus), common carp (C. carpio), and catfish (P. hypophthalmus) which received high concentrations of busulfan (80–120 mg kg−1 and 40 mg kg−1, respectively), showed no lethal effect, suggesting possible species-specific effects of busulfan (Majhi et al., Reference Majhi, Rasal, Kushwaha and Raizada2017, Reference Majhi, Maurya, Kumar, Mohindra and Lal2019; Wang et al., Reference Wang, Liang, Zou, Wang and You2021). In the Patagonian pejerrey (Odontesthes hatcheri), ulcerations and some mortalities were observed in fish that received the highest dose of busulfan (40 mg kg−1), with females being mostly affected (S. K. Majhi et al., Reference Majhi, Hattori, Yokota, Watanabe and Strüssmann2009). In contrast, in the young olive flounder (P. olivaceus), busulfan caused atrophy, discoloration, and adhesion of the visceral mass to the gonads, with males being more frequently affected (Wang et al., Reference Wang, Liang, Zou, Wang and You2021).
The strategy used in experiment 2 combined two sterilization methods. First, chromosomal manipulation was applied to produce sterile triploids of A. altiparanae, which still retain germ cells in the gonads (Ferreira Do Nascimento et al., Reference Ferreira Do Nascimento, De Siqueira-Silva, Pereira-Santos, Fujimoto, Senhorini, Nakaghi and Yasui2017; Piva et al., Reference Piva, de Siqueira-Silva, Goes, Fujimoto, Saito, Dragone, Senhorini, Porto-Foresti, Ferraz and Yasui2018). Based on previous results in diploid of A. altiparanae, triploid juveniles were treated with three applications of busulfan (50 mg kg−1) to deplete spermatogenesis. This approach aimed to remove the germ cells barrier in the testes, facilitating colonization by transplanted cells while delaying the recovery of endogenous spermatogenesis.
This work reported, for the first time, the complete depletion of spermatogenesis in juvenile triploid A. altiparanae by chemical sterilization using busulfan associated with high temperature (35°C). In previous work, spermatogenesis depletion was achieved in diploid A. altiparanae chemically sterilized with busulfan (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, Ninhaus-Silveira and Veríssimo-Silveira2015). In diploid A. altiparanae, reactivation of endogenous spermatogenesis was observed twenty-one days after SSCs stem cell transplantation, with the recipient’s testis presenting endogenous sperm (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, da Silva Costa, Senhorini, Ninhaus-Silveira and Veríssimo-Silveira2019b). Although germ cell transplantation using adult recipients previously treated with busulfan reduces the time required to produce donor-derived gametes, the production of endogenous gametes is disadvantageous for the success of the transplant, as it competes with exogenous cells for space and nutrients, limiting the proliferation of the latter in the recipient gonad (Brinster and Zimmermann, Reference Brinster and Zimmermann1994; Kubota et al., Reference Kubota, Avarbock and Brinster2004).
SSCs transplantation in adults is easy execute, as this technique does not require sophisticated equipment, in contrast to be performed compared to cell transplantation into embryos or larvae, making it a more practical approach (Saito et al., Reference Saito, Goto-Kazeto, Arai and Yamaha2008; Morita et al., Reference Morita, Kumakura, Morishima, Mitsuboshi, Ishida, Hara, Kudo, Miwa, Ihara, Higuchi, Takeuchi and Yoshizaki2012; Hattori et al., Reference Hattori, Yoshinaga, Katayama, Hattori-Ihara, Tsukamoto, Takahashi and Tabata2019). However, performing SSCs transplantation in adult gonads requires recipient have a germ cell-free gonad to allow the transplanted cell to enter the germinal epithelium. Furthermore, it is essential that the recolonization of endogenous cells be delayed or eliminated, allowing for the colonization and multiplication of the transplanted cells. Therefore, alternative strategies must be implemented to generate recipients with germ cell-free gonads, providing favorable conditions for SSCs transplantation and the colonization of the recipient gonads. Among these strategies, increasing the number of chemical sterilizer applications, reducing application intervals, determining the concentration of the sterilizing agent that provides greater effectiveness with lower mortality, and identifying optimal temperature for use with the sterilizing agent could be considered.
In addition, recipients with more physiologic and immunologic compatibility with donors could favor the transplanted cells, as well as the genetic makeup of the recipient, such as diploid, triploid, and hybrid. Some complete sterilization strategies have been successful using busulfan treatment in combination with high temperatures, as in the case of carp (C. carpio) (Majhi et al., Reference Majhi, Rasal, Kushwaha and Raizada2017) and catfish (P. hypophthalmus) (Majhi et al., Reference Majhi, Maurya, Kumar, Mohindra and Lal2019). In the experiments carried out in this study, the animals collected 30 days after the last busulfan application showed only a few SSCs in the germinal epithelium in the non-transplanted control group, indicating the beginning of endogenous spermatogenesis. The sterilization procedures used in this work could be altered to avoid a premature return of endogenous spermatogenesis, such as by increasing the number of busulfan applications. Compared to diploid adults, spermatogenesis is slower in triploid A. altiparanae (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, da Silva Costa, Senhorini, Ninhaus-Silveira and Veríssimo-Silveira2019b), suggesting that the return of spermatogenesis after chemical sterilization in triploid A. altiparanae could be slower than in diploid A. altiparanae. This fact suggests triploid A. altiparanae could be a more efficient recipient than diploid ones treated with chemical sterilization.
The results obtained from the transplantation of B. orbignyanus SSCs into triploid of A. altiparanae treated with busulfan were promising (40.74% efficiency). In this work, the first study using adult triploid receptors treated with busulfan for the transplantation of germ cells was reported. This strategy can be innovative for the generation of germline chimeras. Germ cell PKH26-positive cells were identified in the gonads of recipients up to 80 days after transplantation. In the previous work in which B. orbignyanus SSCs were transplanted into gonads of diploid male A. altiparanae recipients, spermatozoa were observed 21 days after transplantation (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, da Silva Costa, Senhorini, Ninhaus-Silveira and Veríssimo-Silveira2019b). In other studies, using chemical sterilization with busulfan, spermatogenesis was depleted, and endogenous spermatozoa were also visualized (Sulip K. Majhi et al., Reference Majhi, Hattori, Rahman, Suzuki and Strüssmann2009; NÓBREGA et al., Reference Nóbrega, Greebe, van de Kant, Bogerd, de França and Schulz2010; Lacerda et al., Reference Lacerda, Batlouni, Costa, Segatelli, Quirino, Queiroz, Kalapothakis and França2010; Majhi et al., Reference Majhi, Hattori, Rahman and Strüssmann2014; Angel Andreas Arias Vigoya et al., Reference Vigoya, Costa, Oliveira, Butzge, Rosa, Doretto, Martinez, Digmayer and Nóbrega2024). Although in our work no gametes were collected from animals (n = 6), the results obtained serve as a starting point to carry out further work and find the most opportune moment to carry out cell transplantation into adult triploid fish. Published studies on germ cells transplantation into embryos, larvae, or adults have shown that few individuals are capable of producing donor gametes, with greater success almost always observed in intraspecific transplants (Okutsu et al., Reference Okutsu, Shikina, Kanno and Takeuchi2007; Lacerda et al., Reference Lacerda, Batlouni, Costa, Segatelli, Quirino, Queiroz, Kalapothakis and França2010; Hattori et al., Reference Hattori, Yoshinaga, Katayama, Hattori-Ihara, Tsukamoto, Takahashi and Tabata2019), and limited success in transplantation at the interspecific level (Sulip K. Majhi et al., Reference Majhi, Hattori, Rahman, Suzuki and Strüssmann2009; Morita et al., Reference Morita, Morishima, Miwa, Kumakura, Kudo, Ichida, Mitsuboshi, Takeuchi and Yoshizaki2015; Zhou et al., Reference Zhou, Wang, Liu, Yang, Xu, Wu, Wang, You, Song and Li2021). Studies suggest that immunosuppressants, such as tacrolimus, may be promising to prevent cell rejection in testicular allograft transplantation or germ cell transplantation, being an alternative to optimize the immunosuppressive effects in host organisms, as was done in rainbow trout (Oncorhynchus mykiss) (Yoshinaga et al., Reference Yoshinaga, Kfoury Júnior, Butzge, Olio, Hernandez-Blazquez, Oliveira Carreira, de Oliveira Massoco Salles Gomes, Bianchi, Tabata and Hattori2021).
Although A. altiparanae has been used as a recipient of SSC from B. orbignyanus, either using busulfan-treated diploid adult males (de Siqueira-Silva et al., Reference de Siqueira-Silva, dos Santos Silva, da Silva Costa, Senhorini, Ninhaus-Silveira and Veríssimo-Silveira2019b) or triploid hybrid adult males (López et al., Reference López, Monzani, Carvalho, de Siqueira Silva, Vianna, Yasui and Senhorini2024), it has never been possible to obtain gametes from the donor species. In males treated with busulfan, only a few spermatozoa were observed in the testicular lumen, but no spermatozoa were collected for further evaluation. In triploid hybrid males, although sperm were collected, they did not present motility, and molecular evaluations did not show expression of the donor species (López et al., Reference López, Monzani, Carvalho, de Siqueira Silva, Vianna, Yasui and Senhorini2024). Although A. altiparanae is physiologically similar to B. orbignyanus, they are phylogenetically distant, which may be a limiting factor for obtaining gametes. Similar results were reported by Blanes-García et al. (Reference Blanes-García, Marinović, Morini, Vergnet, Horváth and Asturiano2024) using European Eel (Anguilla anguilla) as donor species and zebrafish (Danio rerio) and European Sea Bass (Dicentrarchus labrax) as recipient species. In both recipient species, SSCs of the donor species were not detected in the gonads of the recipients one and a half months after transplantation. Additionally, molecular evaluations did not detect expression of the donor species. Similar results were observed in interspecific transplants between Jundia catfish (Rhamdia quelen) into adult Nile tilapia (O. niloticus) (Silva et al., Reference Silva, Costa, Lacerda, Brandão-Dias, Kalapothakis, Silva Júnior, Alvarenga and França2016) and between Jundia catfish (R. quelen) into adult Common Carp (C. carpio) (Rosa et al., Reference Rosa, Martinez, Digmayer, Doretto and Nóbrega2023). In this process, although cell colonization and proliferation were demonstrated in the recipient’s gonad, it was not possible to collect gametes. Therefore, the results suggest that the gonadal microenvironments are inadequate for spermatogenesis development in the donor species.
Although it was not possible to collect B. orbignyanus gametes using triploid A. altiparanae recipients treated with busulfan, the established procedures could serve as a basis for depleting spermatogenesis in other triploid species such as Brycon amazonicus (Do Nascimento et al., Reference Do Nascimento, Bertolini, Lopez, Nakaghi, Monzani, Senhorini, Vianna and Yasui2021) or Prochilodus lineatus (Yasui et al., Reference Yasui, Nakaghi, Monzani, Nascimento, Pereira dos Santos, Goes, Porto-Foresti and Senhorini2020), species that are phylogenetically and physiologically closer to B. orbignyanus. Some studies have shown that only some triploid males produce sperm, presenting low fertilization rates and not obtaining viable offspring (Ferreira Do Nascimento et al., Reference Ferreira Do Nascimento, De Siqueira-Silva, Pereira-Santos, Fujimoto, Senhorini, Nakaghi and Yasui2017; Takeuchi et al., Reference Takeuchi, Yatabe, Yoshikawa, Ino, Kabeya, Yazawa and Yoshizaki2018). This fact prevents the hybridization of descendants of the donor and recipient species (Sulip K. Majhi et al., Reference Majhi, Hattori, Rahman, Suzuki and Strüssmann2009; Lacerda et al., Reference Lacerda, Batlouni, Costa, Segatelli, Quirino, Queiroz, Kalapothakis and França2010). Triploid animals can develop normal gametogenesis when they are used as germ cell receptors, either in the stages of embryos or larvae, reaching the point of producing gametes only from the donor individual (Saito et al., Reference Saito, Goto-Kazeto, Arai and Yamaha2008; Yoshizaki et al., Reference Yoshizaki, Ichikawa, Hayashi, Iwasaki, Miwa, Shikina and Okutsu2010; Iwasaki-Takahashi et al., Reference Iwasaki-Takahashi, Shikina, Watanabe, Banba, Yagisawa, Takahashi, Fujihara, Okabe, Valdez, Yamauchi and Yoshizaki2020). The results obtained so far indicate that the strategy of using busulfan-treated A. altiparanae triploids may be a promising for SSCs transplantation.
Conclusion
The complete depletion of germ cells in triploid juveniles of A. altiparanae was obtained for the first time in this work through the combination between triploidization and chemical sterilization. Transplantation via the urogenital papilla made it possible to visualize the colonization of the transplanted cells in the male recipients up to 80 days after transplant. The work on sterilization and transplantation of cells for adult animals carried out here, is of great value for the generation of piracanjuba germline chimeras to be used as conservation measures and species reconstitution.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0967199426100380.
Acknowledgements
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 conflict of interest.
Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.
