Aquaculture, cryopreservation, biotechniques, fish reproduction
12
Scopus Publications
61
Scholar Citations
5
Scholar h-index
4
Scholar i10-index
Scopus Publications
A stepwise methodology for developing fish sperm cryopreservation protocols: application to the endangered Pogonias courbina Jhony Lisbôa Benato, Nathalia dos Santos Teixeira, Rômulo Batista Rodrigues, Karel Torres Lozano, Renata Villar Dantas, Eduardo Thomé Nicoleti, Gregory Valdivia-Gutiérrez, Leonardo Queiroz de Alencar, Wik Maik Santos Barros, Marcelo Hideo Okamoto, Ricardo Vieira Rodrigues, Luis Andre Sampaio, Danilo Pedro Streit Cryobiology, 2026 Wild fish populations face growing threats from anthropogenic activities, climate change, and natural disasters. Aquatic germplasm banking is a vital strategy for preserving genetic diversity and supporting sustainable aquaculture. This study proposes a sequential, decision-guided framework for developing species-specific sperm cryopreservation protocols, applied here to the endangered Pogonias courbina, listed as endangered in Brazil and vulnerable by the IUCN. A series of experiments was conducted to identify optimal conditions for thawing (26 °C for 14 s), equilibration time (10 min), extenders (HBSS, 3% glucose, 5% glucose), and both permeable (dimethyl sulfoxide, propylene glycol, dimethylformamide, ethylene glycol) and non-permeable cryoprotectants (glucose, fructose, sucrose, trehalose, skimmed powdered milk, egg yolk). Sperm quality was assessed using Computer-Assisted Sperm Analysis (CASA). Three treatments with the highest post-thaw motility (MOT) were selected to fertilize oocytes from two hormonally-induced females. No significant difference was observed between sperm from wild and farmed males. The best kinetic parameters and fertilization rates were obtained using the combination of HBSS +10% DMSO +5% egg yolk (MOT: 71.8%). Based on these findings, we propose practical recommendations regarding extender selection, equilibration time, cryoprotectant combinations, and thawing conditions that support high post-thaw viability and successful fertilization. This is the first study to report successful artificial reproduction of P. courbina using cryopreserved sperm, establishing a reproducible protocol that can be applied for captive breeding, restocking, and germplasm banking. The proposed framework offers a generalizable approach for developing cryopreservation protocols in fish species.
Reproductive biology characterization of violinha Loricariichthys anus Douglas C. Selle, Raquel S. dos Santos, Thaiza R. de Freitas, Jhony L. Benato, Thales de S. França, Renata V. Dantas, Marco A. Rotta, Marcelle F.O. Barbosa, Vanessa Coimbra, Diógenes H. Siqueira-Silva, Danilo P. Streit Reproductive Biology, 2026
Celiotomy to collect spermatogonial stem cells in Brycon orbignyanus for species preservation Laís Gonçalves da Silva, Karel Gelina Torres-Lozano, Rômulo Batista Rodrígues, Thaiza Rodrigues de Freitas, Jayme Aparecido Povh, Louise Nex Spica, Nathalia dos Santos Teixeira, Douglas Cosme Selle, Jhony Lisbôa Benato, Raquel Santos dos Santos, Eduardo Thomé Nicoleti, Renata Villar Dantas, Danilo Pedro Streit Junior Animal Reproduction, 2026 Surgical techniques in aquatic species are underdeveloped, despite these species comprising a significant superclass among vertebrates. In the context of species preservation, studies involving the use of reproductive tissues typically involve the euthanasia of donors. Thus, in animals at high risk of extinction, the practice of sacrificing them presents a point of contradiction between ex situ conservation efforts. The objective of this study was to compare the viability of spermatogonia stem cells (SSCs) collected from Brycon orbignyanus using conventional methods (euthanasia) versus a surgical procedure. Lateral celiotomy was performed on 27 immatures males to obtain a portion of gonadal tissue. The fish were divided into three groups (n=9), with each group receiving polyglactin 910, polyester, or catgut sutures, respectively, for celiorrhaphy. Dermorraphy was performed using nylon sutures in all groups. A. An additional euthanasia group consisted of nine animals exposed to 20 mg/L of propofol. The survival rate over a 48-hour period was 100% for the polyglactin 910 group and 77.7% for the polyester and catgut groups. Additionally, the viability of SSCs was similar between the euthanasia and surgical procedures. The lateral celiotomy technique is feasible for obtaining SSCs in fish with laterally compressed anatomy, such as B. orbignyanus. Additionally, the technique allows the preservation of SSCs as a model for endangered fish species.
Tricaine methanesulfonate and eugenol during semen collection do not affect fertilization and hatching rates on Rhamdia quelen Nathalia dos Santos Teixeira, Lis Santos Marques, Rômulo Batista Rodrigues, Thales de Souza França, Jhony Lisbôa Benato, Maritza Pérez Atehortúa, Darlan Gusso, Itamar Cossina Gomes, Thaiza Rodrigues de Freitas, Henrique Mautone Gomes, Helen Tais Rosa-Silva, Alexandre Kleber Silveira, José Claudio Fonseca Moreira, Danilo Pedro Streit Junior Animal Reproduction, 2026 The reproduction of some fish species in captivity is only possible by imposing artificial reproductive procedures, and the manipulation of fish for these purposes is a stressor. Thus, anesthesia can reduce stress during handling. However, it is necessary to investigate the possible side effects on breeding and the general health of the animal. Therefore, we aimed to investigate the impact of tricaine methanesulfonate (MS-222) and eugenol (EUG) at concentrations of 300 mg L-1 and 40 mg L-1, respectively, on several stress- and reproductive-related parameters in Rhamdia quelen anesthetized before semen collection. After hormonal induction, 24 sexually mature males (534.4 ± 259 g) were randomly assigned to treatment groups and semen and blood samples were collected. Anesthesia recovery and induction time were extended in fish anesthetized with EUG; however, plasma cortisol concentrations did not differ among the treatment groups. EUG caused higher DNA fragmentation in blood cells than in the control group (without anesthesia). EUG increased the monocyte count compared to the other experimental groups. MS-222 showed a lower sulfhydryl group (SH) quantitation than the other experimental groups. The anesthetics used before semen collection in this study had no adverse effects on the fertilization or hatching capacity of anesthetized R. quelen. MS-222 (300 mg L-1) and EUG (40 mg L-1), despite being related to hematological and semen changes, did not negatively affect the reproductive capacity of R. quelen males. Thus, anesthesia is recommended for R. quelen during semen collection, considering reproductive parameters and animal welfare.
A Systematic Review of Brycon Genus Sperm Cryopreservation Protocols: Challenges on Ex-Situ Conservation for Sustainable Aquaculture Danilo Pedro Streit, Rômulo Batista Rodrigues, Jhony Lisboa Benato, Gregory Brayan Valdívia‐Gutiérrez, Eduardo Thomé Nicoleti, Leonardo Queiroz de Alencar, Thales Lysakowski Flores Machado, Larise Caroline Oliveira Lima, Marco Aurélio Rotta, Thales de Souza França Reviews in Aquaculture, 2025 This systematic review shows an overview of the history of protocols used for genus Brycon sperm cryopreservation. Relevant studies were identified from Web of Science, Scopus, PubMed, and Scielo databases. Thirty‐four studies published between 2001 and 2021 were included in the systematic review following the eligibility criteria. Selected studies showed cryopreservation protocols of nine species of the genus Brycon, with Brycon orbignyanus being the most studied species. The studies were conducted by Brazilian (76.5%) and Colombian (23.5%) researchers, with the majority (68.6%) carried out in hydroelectric plants promoting fish restocking programs. Two main protocols were extensively investigated across nine studied species: 10% methyl glycol +5% Beltsville Thawing Solution and 10% dimethyl sulfoxide +5% glucose. However, the protocols lacked standardization regarding equilibrium time and temperature, freezing and thawing curves, or sperm‐to‐cryoprotectant solution ratios. Limited collaboration among research groups from the two South American countries was identified as a significant factor in this lack of standardization. In addition, emerging approaches for sperm cryopreservation are necessary, such as developing less toxic alternative cryoprotectants. Implementing advanced cryopreservation methodologies and using artificial intelligence and machine learning can optimize protocols by modeling key parameters and enhancing post‐thaw sperm quality. These findings underscore the need for collaboration between research groups to develop standardized cryopreservation protocols and establish functionally accessible germplasm banks. Such measures are essential for the ex‐situ conservation and long‐term sustainability of aquaculture involving species of the genus Brycon.
Understanding Rigor Mortis Impacts on Zebrafish Gamete Viability Renata Villar Dantas, Lis Santos Marques, Thaiza Rodrigues Freitas, Nathalia dos Santos Teixeira, Rômulo Batista Rodrigues, Jhony Lisbôa Benato, Raquel Santos dos Santos, Diógenes Henrique Siqueira Silva, Danilo Pedro Streit Zebrafish, 2025 This study aimed to evaluate the viability of gametes in zebrafish (Danio rerio), at different rigor mortis stages. Viability assessments were conducted on oocytes at various developmental stages using LIVE/DEAD and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. For sperm evaluation, both kinetic (computer-assisted sperm analysis) and morphological assessments (Rose Bengal staining) were performed. Results demonstrated that rigor mortis progression significantly impacted oocyte viability during post-rigor stages, with the following viability rates: pre-rigor (70.43 ± 12.31%), fresh/control (46.43 ± 12.54%), post-rigor (27.62 ± 22.29%), and rigor mortis (comparable to fresh/control). Conversely, sperm kinetics exhibited nuanced responses to the rigor mortis stages, with specific parameters showing sensitivity, whereas the others remained relatively stable. Sperm motility was higher in the fresh/control (63.23 ± 19.03%) and pre-rigor (58.96 ± 14.38%) compared to the post-rigor group (3.34 ± 4.65%). This study highlights the significance of the pre-rigor for successful gamete collection and preservation. These findings provide valuable insights for conservation efforts and optimization of genetic resource management for endangered fish species. This study aimed to develop effective assistive reproductive techniques by elucidating the interplay between rigor mortis and gamete quality, contributing to the broader goals of species conservation and maintenance of genetic diversity in fish populations.
Potential Use of Germplasm Banks of Neotropical South American Species for Fish Farming in Brazil Danilo P. Streit, Jayme A. Povh, Darci C. Fornari, Jonhy L. Benato, Fernanda De Mello, Eric A. B. Routledge, Estefania Paredes, Tiantian Zhang Cryobiology for South American Neotropical Fish Species, 2024 Brazil is one of the main players in global food production (both vegetables and animals). Even though Brazil is not currently one of the world’s leading fish producers, the activity has grown steadily over the last ten years. However, according to the FAO, the future of aquaculture sustainability is consistently linked to the diversity of species that must be produced. In this context, Germplasm Banks can and are crucial in developing and consolidating different South American species with significant potential. The National Germplasm Bank plays a crucial role in developing Brazilian fish farming using native species.
Laboratory Techniques for the Analysis of Cells and Reproductive Tissues of Neotropical Species Lauren Dorneles Dipp, Lis Santos Marques, Rômulo Batista Rodrigues, Thales de Souza França, Nathalia dos Santos Teixeira, Thaiza Rodrigues De Freitas, Raquel Santos Dos Santos, Larise Lima, Jhony Lisboa Benato, Danilo P. Streit Cryobiology for South American Neotropical Fish Species, 2024 The knowledge about the current methods and the dissemination of the new ones contribute to developing the research in many different areas. Especially when a standardization protocol is applied to a new biomodel species, the biology and specific parameters need attention. In this chapter, we bring the main methods used in the AQUAM laboratory to evaluate fish reproductive cells (sperm cells and eggs) quality and viability after and before being submitted to cryopreservation treatments. The research group aims for the management and conservation of South American native fish species, so these methods are widely applied to different fish species, and some of them must be available to use outside the laboratory in the field. We believe the spread of these methods will contribute to other laboratories and even help to improve these techniques in future studies.
Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology Thales de Souza França, Itamar Cossina Gomes, Eduardo Antônio Sanches, Maritza Pérez Atehortúa, Nathalia dos Santos Teixeira, Rômulo Batista Rodrigues, Thaiza Rodrigues de Freitas, Jhony Lisbôa Benato, Lis Santos Marques, Ana Regina Seabra de Souza, Danilo Pedro Streit Reproduction, 2023 In brief The containers used in cell cryopreservation are essential to maintain cell integrity and viability after thawing. This paper reveals the methodology of using biodegradable containers for fish sperm cryopreservation. Cryopreserved sperm in biodegradable containers showed high fertility capability. Biodegradable capsules could be alternative containers to plastic straws for sperm cryopreservation. Abstract Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from 12South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of the tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.
Pogonias courbina sperm characteristcs in its first reproductive season Jhony Lisboa Benato, Danilo Streit Jr, Nathalia Dos Santos Teixeira, Rômulo Batista Rodrigues, Thaiza Rodrigues de Freitas, Marcelo Okamoto, Ricardo Rodrigues, Raquel Santos dos Santos, Renata Villar Dantas, Ana Paula de Abreu Balbinot, Rodrigo Ribeiro Bezerra de Oliveira, Lucas Campos Maltez, Olivia Menossi, Luis Andre Sampaio Peerj, 2023
Post-thaw dilution of Rhamdia quelen sperm improves the reproductive success Thales de Souza França, Itamar Cossina Gomes, Eduardo Antônio Sanches, Maritza Pérez Atehortúa, Nathalia Santos Teixeira, Rômulo Batista Rodrigues, Thaiza Rodrigues de Freitas, Andrea Giannotti Galuppo, Monike Quirino, Jhony Lisbôa Benato, Thales Lysakowski Flores Machado, Lis Santos Marques, Ivan Cunha Bustamante-Filho, Fernando Pandolfo Bortolozzo, Danilo Pedro Streit Jr Animal Reproduction Science, 2022
RECENT SCHOLAR PUBLICATIONS
A stepwise methodology for developing fish sperm cryopreservation protocols: application to the endangered Pogonias courbina JL Benato, N dos Santos Teixeira, RB Rodrigues, KT Lozano, RV Dantas, ... Cryobiology 123, 105604 , 2026 2026
Tricaine methanesulfonate and eugenol during semen collection do not affect fertilization and hatching rates on Rhamdia quelen NS Teixeira, LS Marques, RB Rodrigues, TS França, JL Benato, ... Animal Reproduction 23 (2), e20240107 , 2026 2026
Celiotomy to collect spermatogonial stem cells in Brycon orbignyanus for species preservation LG Silva, KG Torres-Lozano, RB Rodrígues, TR Freitas, JA Povh, ... Animal Reproduction 23 (1), e20250085 , 2026 2026
Reproductive biology characterization of violinha Loricariichthys anus DC Selle, RS Dos Santos, TR de Freitas, JL Benato, TS França, ... Reproductive Biology 26 (1), 101171 , 2026 2026
Cryopreservation protocols in genus Brycon: Strategic tools for conservation and sustainable aquaculture DP Streit, J Benato, G Valdivia, ET Nicoleti, L Alencar, T Flores, L Lima, ... Cryobiology 121, 105420 , 2025 2025
Enhancing aquaculture efficiency: Development of a reliable cryopreservation protocol for leiarius marmoratus ET Nicoleti, RB Rodrigues, JL Benato, ND Santos Teixeira, LN Spica, ... Cryobiology 121, 105432 , 2025 2025
Establishing a sperm cryopreservation protocol: Application to the endangered Pogonias courbina JL Benato, NS Teixeira, RB Rodrigues, KT Lozano, RV Dantas, ... Cryobiology 121, 105424 , 2025 2025
A Systematic Review of Brycon Genus Sperm Cryopreservation Protocols: Challenges on Ex‐Situ Conservation for Sustainable Aquaculture DP Streit Jr, RB Rodrigues, JL Benato, GB Valdívia‐Gutiérrez, ET Nicoleti, ... Reviews in Aquaculture 17 (4), e70064 , 2025 2025 Citations: 1
Methods for greenhouse gas emission accounting in inland aquaculture: a systematic review SAF Streit, MA Rotta, JL Benato, RB Rodrigues, Ú da Silva Morales, ... Environmental Monitoring and Assessment 197 (6), 644 , 2025 2025
Understanding Rigor Mortis Impacts on Zebrafish Gamete Viability RV Dantas, LS Marques, TR Freitas, NS Teixeira, RB Rodrigues, ... Zebrafish 22 (2), 31-41 , 2025 2025
Cryobiology for South American Neotropical Fish Species DP Streit, T Zhang, EP Rosendo CRC Press , 2024 2024 Citations: 5
Laboratory Techniques for the Analysis of Cells and Reproductive Tissues of Neotropical Species LD Dipp, LS Marques, RB Rodrigues, T de Souza França, ... Cryobiology for South American Neotropical Fish Species, 183-192 , 2024 2024
Insecticides Cypermethrin and Chlorpyrifos Exposure: Effects on Sperm Motility and Fertility in Cyprinus carpio (Cypriniformes, Cyprinidae) F Argemi, AA Porta, D Streit Jr, RB Rodrigues, JL Benato, FL Nostro Ecotoxicology and Environmental Contamination 19 (1), 20-29 , 2024 2024
Caracterização espermática e criopreservação seminal de Pogonias courbina JL Benato 2024
Descriptive analysis of the sperm of three South American catfish species maintained ex situ and the influence of morphology on sperm movement BB da Costa, RB Rodrigues, JA Povh, EA Sanches, ... Theriogenology Wild 4, 100080 , 2024 2024
Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology T de Souza França, IC Gomes, EA Sanches, MP Atehortúa, ... Reproduction 166 (2), 89-97 , 2023 2023 Citations: 12
Pogonias courbina sperm characteristcs in its first reproductive season JL Benato, D Streit Jr, NDS Teixeira, RB Rodrigues, TR de Freitas, ... PeerJ 11, e15600 , 2023 2023 Citations: 4
Post-thaw dilution of Rhamdia quelen sperm improves the reproductive success T de Souza França, IC Gomes, EA Sanches, MP Atehortúa, NS Teixeira, ... Animal Reproduction Science 243, 107018 , 2022 2022 Citations: 10
The use of differential separation and density gradient with AllGrad 90% after thawing improves the sperm quality of South American catfish (Rhamdia quelen) M Pérez-Atehortúa, AG Galuppo, RB Rodrigues, T de Souza França, ... Aquaculture 553, 738072 , 2022 2022 Citations: 15
Desenvolvimento de protocolo de criopreservação espermática de Miraguaia (Pogonias courbina) para formação de banco de germoplasma da espécie JL Benato 2022
MOST CITED SCHOLAR PUBLICATIONS
The use of differential separation and density gradient with AllGrad 90% after thawing improves the sperm quality of South American catfish (Rhamdia quelen) M Pérez-Atehortúa, AG Galuppo, RB Rodrigues, T de Souza França, ... Aquaculture 553, 738072 , 2022 2022 Citations: 15
Characterization of sperm quality in Brycon hilarii: How does morphology affect sperm movement? BB da Costa, JA Povh, EA Sanches, LN Spica, RB Rodrigues, ... Theriogenology Wild 1, 100007 , 2022 2022 Citations: 13
Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology T de Souza França, IC Gomes, EA Sanches, MP Atehortúa, ... Reproduction 166 (2), 89-97 , 2023 2023 Citations: 12
Post-thaw dilution of Rhamdia quelen sperm improves the reproductive success T de Souza França, IC Gomes, EA Sanches, MP Atehortúa, NS Teixeira, ... Animal Reproduction Science 243, 107018 , 2022 2022 Citations: 10
Cryobiology for South American Neotropical Fish Species DP Streit, T Zhang, EP Rosendo CRC Press , 2024 2024 Citations: 5
Pogonias courbina sperm characteristcs in its first reproductive season JL Benato, D Streit Jr, NDS Teixeira, RB Rodrigues, TR de Freitas, ... PeerJ 11, e15600 , 2023 2023 Citations: 4
A Systematic Review of Brycon Genus Sperm Cryopreservation Protocols: Challenges on Ex‐Situ Conservation for Sustainable Aquaculture DP Streit Jr, RB Rodrigues, JL Benato, GB Valdívia‐Gutiérrez, ET Nicoleti, ... Reviews in Aquaculture 17 (4), e70064 , 2025 2025 Citations: 1
NaCl Extender Improves Kinetics Parameters and Reproductive Capacity of Fish Post-Thaw Sperm TS França, IG Cossina, R Rodrigues, MP Atehortúa, NS Teixeira, ... Cryobiology 103, 188 , 2021 2021 Citations: 1
A stepwise methodology for developing fish sperm cryopreservation protocols: application to the endangered Pogonias courbina JL Benato, N dos Santos Teixeira, RB Rodrigues, KT Lozano, RV Dantas, ... Cryobiology 123, 105604 , 2026 2026
Tricaine methanesulfonate and eugenol during semen collection do not affect fertilization and hatching rates on Rhamdia quelen NS Teixeira, LS Marques, RB Rodrigues, TS França, JL Benato, ... Animal Reproduction 23 (2), e20240107 , 2026 2026
Celiotomy to collect spermatogonial stem cells in Brycon orbignyanus for species preservation LG Silva, KG Torres-Lozano, RB Rodrígues, TR Freitas, JA Povh, ... Animal Reproduction 23 (1), e20250085 , 2026 2026
Reproductive biology characterization of violinha Loricariichthys anus DC Selle, RS Dos Santos, TR de Freitas, JL Benato, TS França, ... Reproductive Biology 26 (1), 101171 , 2026 2026
Cryopreservation protocols in genus Brycon: Strategic tools for conservation and sustainable aquaculture DP Streit, J Benato, G Valdivia, ET Nicoleti, L Alencar, T Flores, L Lima, ... Cryobiology 121, 105420 , 2025 2025
Enhancing aquaculture efficiency: Development of a reliable cryopreservation protocol for leiarius marmoratus ET Nicoleti, RB Rodrigues, JL Benato, ND Santos Teixeira, LN Spica, ... Cryobiology 121, 105432 , 2025 2025
Establishing a sperm cryopreservation protocol: Application to the endangered Pogonias courbina JL Benato, NS Teixeira, RB Rodrigues, KT Lozano, RV Dantas, ... Cryobiology 121, 105424 , 2025 2025
Methods for greenhouse gas emission accounting in inland aquaculture: a systematic review SAF Streit, MA Rotta, JL Benato, RB Rodrigues, Ú da Silva Morales, ... Environmental Monitoring and Assessment 197 (6), 644 , 2025 2025
Understanding Rigor Mortis Impacts on Zebrafish Gamete Viability RV Dantas, LS Marques, TR Freitas, NS Teixeira, RB Rodrigues, ... Zebrafish 22 (2), 31-41 , 2025 2025
Laboratory Techniques for the Analysis of Cells and Reproductive Tissues of Neotropical Species LD Dipp, LS Marques, RB Rodrigues, T de Souza França, ... Cryobiology for South American Neotropical Fish Species, 183-192 , 2024 2024
Insecticides Cypermethrin and Chlorpyrifos Exposure: Effects on Sperm Motility and Fertility in Cyprinus carpio (Cypriniformes, Cyprinidae) F Argemi, AA Porta, D Streit Jr, RB Rodrigues, JL Benato, FL Nostro Ecotoxicology and Environmental Contamination 19 (1), 20-29 , 2024 2024
Caracterização espermática e criopreservação seminal de Pogonias courbina JL Benato 2024
