Cryopreservation toxicity and morphological outcomes in Piaractus brachypomus oocytes and embryos Melanie Digmayer, Darci Carlos Fornari, Lis Santos Marques, Jayme Aparecido Povh, Ricardo Pereira Ribeiro, et al. Animal Reproduction, 2026 This study evaluated the toxicity of cryoprotectants and the performance of low-rate freezing protocols for oocytes and embryos of Piaractus brachypomus, a Neotropical fish of increasing relevant for Brazilian aquaculture and genetic conservation. Eight cryoprotectant solutions based on methanol (MeOH) or dimethyl sulfoxide (Me2SO), combined with 0.25 M sucrose in L-15 or HBSS media, were tested for oocytes toxicity at 28 °C and post-freezing viability. In spite of histological and scanning electron microscopy (SEM) analyses indicated preservation of gross morphological features, none of the cryopreserved oocytes supported embryonic development, indicating loss of functional viability following cryoprotectant exposure and freezing. For embryos, two low-rate freezing protocols were evaluated: Protocol 1 (P1E), based on gradual cooling to −13 °C, and Protocol 2 (P2E), involving linear cooling to −60 °C followed by storage in liquid nitrogen. In P1E, eight treatments using 3.1 M MeOH combined with different concentrations of polyvinylpyrrolidone (PVP) or sucrose were tested. The highest proportion of morphologically viable embryos (15.3%) was obtained with MeOT + 0.45 M sucrose (SC5), which different significantly from the other treatments. In contrast, no morphologically viable embryos were recovered after P2E, likely due to inadequate dehydration and intracellular ice formation. Overall, high concentrations of permeant cryoprotectants and prolonged equilibration times were detrimental to oocyte and embryo viability, while sucrose showed better cryoprotective performance than PVP. Even though protocols tested were insufficient to ensure consistent post-thaw viability, the partial success observed in P1E under MeOH and sucrose combinations provides a relevant experimental basis for future refinement of conservation strategies and contributes to development of ex situ germoplasm conservation approaches for P. brachypomus and other Neotropical species.
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, et al. 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.
First Frozen Repository for Coral Sperm in the Southwestern Atlantic: A Tool to Support Reef Conservation Nayara Oliveira da Cruz, Andrea Giannotti Galuppo, Allison Gonçalves Silva, Luciano da Silva Lima, Romulo Batista Rodrigues, et al. Aquatic Conservation Marine and Freshwater Ecosystems, 2025 Coral reefs are among the most biodiverse ecosystems on Earth, yet they are rapidly declining due to climate change and other anthropogenic stressors. Population reductions threaten sexual reproduction in sessile coral species, as increased distances between colonies hinder gamete encounters. Once populations fall below critical density thresholds, natural recovery may become impossible even after stressors are removed. Biobanking of coral germplasm has therefore emerged as a key strategy to preserve genetic diversity and support reef restoration. This study developed and validated a cryopreservation protocol for sperm of the Brazilian endemic reef‐building coral Mussismilia harttii , representing a milestone in coral conservation for the Southwestern Atlantic. Spermatozoa were exposed to dimethyl sulfoxide (DMSO) and methanol (MeOH) at concentrations of 10%, 15% and 20% and subjected to controlled slow freezing and ultra‐rapid freezing. Postthaw analyses assessed motility, mitochondrial activity and viability. The best results were obtained with 20% DMSO and controlled slow freezing, yielding 29.7% ± 0.84% motility and 83.6% ± 2.2% viability. Mitochondrial activity (MTT assay) was higher with 15% MeOH (1.63 ± 0.17) than with DMSO (0.74 ± 0.17). Cryopreserved sperm (20% DMSO + slow freezing) achieved a 100% fertilization rate, statistically equivalent to fresh sperm ( p = 0.4533). This optimized protocol enabled the establishment of the first coral sperm repository in the South Atlantic, currently storing 2.4 billion viable M. harttii spermatozoa. This repository provides a crucial resource to safeguard genetic diversity, strengthen assisted breeding programs and enhance the long‐term conservation and restoration capacity of Brazilian coral reefs.
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, et al. 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, et al. 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.
Cryopreservation-Induced Morphological Changes in Freshwater Fish Sperm: A Systematic Review Bruna Bitencourt da Costa, Paula Graziela Lassen, Danilo Pedro Streit Biopreservation and Biobanking, 2024 A systematic review was performed to summarize the scientific evidence and critically evaluate the effects of cryopreservation on sperm morphology in freshwater fish, and to assess the methodologies for sperm morphology classification. The search strategy was applied to four electronic databases (CAB Direct, Pub Med, Scopus, and ISI Web of Science). The main inclusion criteria involved studies on semen from freshwater fish subjected to the cryopreservation process and evaluation of sperm quality through morphology. The risk of bias was assessed with respect to randomization, allocation concealment, blinding, incomplete outcome data, and selective reporting. A total of 6 publications reporting sperm cryopreservation from 4 species with a total 74 fish individuals were included in this review. A high methodological variability among the results of the studies was observed due to the species-specific protocols and diversity of freshwater fish species studied. All included studies reported negative effects of cryopreservation on sperm quality, especially morphology, highlighting the increase in incidence of sperm abnormalities. However, only five studies statistically compared abnormalities between groups (fresh and cryopreserved sperm). Our results suggest the need to elaborate on a new morphological classification of fish spermatozoa, by considering the structure and physiology of fish sperm. This classification should be developed based on the sperm characterization and observing damage caused by different cryopreservation protocols.