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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 (1 M) 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, although the number of males used for the experiment was borderline, the procedures established here can be applied in future actions for the conservation and reconstitution of the piracanjuba in case of extinction., Competing Interests: Declarations 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. Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Spermatogonia cryopreservation can be a strategy for future conservation actions. The neotropical Siluriformes Pseudopimelodus mangurus was already classified as vulnerable on the Red List of Threatened Species. P. mangurus spermatogonial cells were isolated, assessed, and cryopreserved. Fragments of the testis were enzymatically dissociated, purified using Percoll density gradient, and submitted to differential plating. Fractionated cells were evaluated by microscopy, ddx4 (vasa) relative expression, and alkaline phosphatase activity. Cryopreservation was conducted using ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and propanediol at 1 M, 1.5 M, and 2 M. Cell viability was evaluated and cell concentration was determined. Cell fractions from 20 % and 30 % Percoll gradient bands showed the highest concentrations of spermatogonia. The fraction mix showed 54 % purity and 93 % viability. After differential plating, 60 % purity and 92 % viability were obtained. Spermatogonial cells showed high alkaline phosphatase activity compared to spermatocytes and spermatids. The relative spermatogonial ddx4 expression from the Percoll density gradient was about twice as high as in samples from the testis and the differential plating. The increased ddx4 expression indicated the enrichment of spermatogonial cells by density gradient step and dead cells expressing ddx4 in differential plating, or ddx4 decreasing expression during cell culture. For this reason, cells from the Percoll gradient were chosen for cryopreservation. Propanediol at 1 M demonstrated the best condition for spermatogonial cell cryopreservation, presenting 98 % viability, while dimethylacetamide at 2 M represented the least favorable condition, with approximately 47 % viability. These findings are essential for P. mangurus spermatogonial cell cryopreservation, aiming to generate a spermatogonia cryobank for future conservation efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (-20 °C), ultra-low (-80 °C) and cryogenic temperatures (-196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (-20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (-80 °C) and cryogenic (-196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at -80 °C and -196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation., (Copyright © 2020. Published by Elsevier Inc.)

We aimed to vitrify embryos of Prochilodus lineatus in a high-osmolarity cryoprotectant solution, evaluating, after the vitrification-thawing process, their morphological changes. Thus, 240 embryos in the 20-somite phase (20S) were exposed for 20 min to one main internal cryoprotectant solution (1,2-propanediol-PROP), divided into four immersion sequence steps of five minutes each. The first three steps were performed in solutions containing only a main internal cryoprotectant (PROP-2, 3 and 4 M), and the fourth step in a high-osmolarity solution combining internal (PROP + dimethyl sulphoxide-Me 2 SO) and external cryoprotectants (sucrose-SUC). The final concentration of vitrification was PROP 5 M + Me 2 SO 5 M + SUC 0.2 M. During vitrification, the straws exhibited a translucent solid appearance; however, during thawing, their structure became totally opaque and white. After thawing, the embryos suffered an increase in volume and presented morphological changes including protrusions on the surface of the yolk sac, yolk sac rupture, and optical vesicle degradation. On the inside, we observed intercellular spaces and a yolk syncytial layer (YSL) with altered chromatin. Yet, structures such as somites, neural tube, endoderm and epidermis presented cells with a nucleus and integral mitochondria. We conclude that the use of the tested cryoprotectant solution permits the formation of a vitreous solid and preserves part of the cells of the blastoderm. Yet, the heating protocol does not control recrystallization, resulting in the formation of serious morphological anomalies that prevent the preservation of the embryonic unit., (© 2018 Blackwell Verlag GmbH.)

Groups of one hundred Brycon orbignyanus embryos at the stage of blastopore closure were subjected to different cooling protocols. Different combinations and concentrations of cryoprotectants were tested: sucrose, methanol, ethylene glycol and dimethyl sulfoxide (Me2SO); at different temperatures (0.0±2.0°C and 8.0±2.0°C) and refrigeration times (6, 10, 24, 72 and 168 h), with the exception of the positive control (incubation without previous cooling). At the end of each refrigeration time, the embryos were acclimatized, rehydrated and incubated to determine hatching, survival and deformity rates. Morphological analysis of embryos was also carried out. The results showed that temperature and refrigeration time are critical factors for embryo survival. No embryos survived after 24, 72 and 168 h of refrigeration. Furthermore, when the refrigeration time increased from 6 to 10h and the temperature decreased from 8.0±2.0°C to 0.0±2.0°C, mortality rates increased significantly. It was also found that in all protocols dead eggs and/or larvae with some degree of deformity were present. The main larval deformities observed were the malformation of the head, tail, yolk sac, vertebral column and eyes., (Copyright © 2014 Elsevier Inc. All rights reserved.)