ABSTRACT
This study evaluated the effect of disaccharide supplementation on zebrafish sperm stored at 5°C for 24 h. Seven pooled replicates from 60 broodfish were used. Semen samples were initially cooled and, after 1 h, supplemented with either HBSS (E‐C), Sucrose 0.13M (E‐S13), Trehalose 0.13M (E‐T13) or Trehalose 0.26M (E‐T26). Sperm motility (computer‐assisted sperm analysis) and plasma membrane integrity (flow cytometry) were evaluated over 24 h of cool storage. During the first 2 h, E‐T26 decreased sperm motility values. After 24 h, E‐S13 and E‐T13 showed higher motility than E‐C and E‐T26. In conclusion, sucrose and trehalose supplementation improved sperm motility after 24 h of cold storage. Further studies are needed to assess the effect of disaccharide supplementation after shorter storage periods.
Keywords: cooling, fish, spermatozoa, sucrose, trehalose
1. Introduction
Zebrafish ( Danio rerio ) is the aquatic model most widely used in biomedical research worldwide (Yang et al. 2016). Consequently, the optimisation of sperm preservation is crucial to preserve the genetic pool (Cheng et al. 2021). Although sperm cryopreservation enables indefinite time storage of genetic material, this technology is complex, costly, and causes sperm damage (Rodrigues et al. 2021). Sperm cooling is a less harmful alternative that simplifies the exchange of gametes between nearby research centres (Zhang et al. 2025).
However, zebrafish semen cooling remains poorly developed (Yang et al. 2016). Currently, zebrafish semen is stored at 5°C for up to 2 h without cryoprotectants (Cheng et al. 2021). In other species, the addition of non‐permeable cryoprotectants (NPC) has improved sperm parameters during cooling. Disaccharides such as sucrose and trehalose in concentrations from 0.1 to 0.3M have been used in aquaculture to preserve sperm membranes during cooling by buffering osmotic stress and helping stabilise the phospholipids of the sperm membrane (de Almeida‐Monteiro et al. 2025; Contreras et al. 2020; Zhu et al. 2016; Anjos et al. 2021; Zalazar et al. 2024). However, no studies have evaluated the effect of NPC addition to extend the longevity of zebrafish sperm (Félix et al. 2020).
The aim of this work was to evaluate the effect of the addition of sucrose and trehalose to improve sperm quality parameters in cooled zebrafish sperm stored for up to 24 h.
2. Materials and Methods
2.1. Semen Collection and Processing
Wild‐type AB adult male zebrafish were provided by the Córdoba University Animal Experimentation Service and maintained according to the legislation for animal experimentation and welfare.
Semen was collected by stripping from anaesthetised males. After collection, 1 μL of sperm was diluted in 10 μL of Hank's Balanced Salt Solution (HBSS; H9394, Sigma‐Aldrich, Madrid, Spain) at 28°C (Diogo et al. 2018). After that, semen from 20 fish was pooled; a total of 60 broodfish and 7 pools were used.
Fresh pooled samples (Fresh) were cooled (≈5°C) in an Equitainer (Hamilton, Biovet, USA) and transported to the laboratory within 1 h. There, sperm motility of the short‐term cooled sample (S‐Cooled, < 1 h) immediately before the addition of disaccharides was evaluated (Diogo et al. 2018).
S‐Cooled was diluted 1:1 with four extended‐cooling (E‐) storage treatments: Control (HBSS, E‐C), Sucrose 0.13M + 1% BSA (E‐S13), Trehalose 0.13M + 1% BSA (E‐T13) and Trehalose 0.26M + 1% BSA (E‐T26). The final osmolarity of these suspensions was, respectively: 269.0 ± 3.9, 409.5 ± 18.2, 391.3 ± 11.9 and 569.0 ± 10.6 mOsm/Kg (Type 15, Löser Messtechnik, Berlin, Germany).
Semen analysis of E‐samples was performed after 0, 10, 20, 30, 60 and 1440 min (24 h) of cold storage after disaccharide addition (1–24 h).
2.2. Sperm Parameters
Sperm motility was evaluated by computer‐assisted sperm analysis (SCA, Microptic, Barcelona, Spain) (Diogo et al. 2018). The parameters evaluated included total (TM) and progressive motility (PM), and curvilinear velocity (VCL), straight‐line velocity (VSL), average path velocity (VAP), straight‐line velocity (VSL), straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Motility activation was performed adding activation medium at 28°C (distilled water and HBSS) to reach a final osmolality of 170 mOsm/kg; sperm was recorded within 10 s after activation (Zhang et al. 2025).
Plasma Membrane Integrity (PMI) was assessed by flow cytometry (Accuri C6 Plus; BD, Franklin Lakes, NJ, USA) using Propidium Iodide staining (Diogo et al. 2018). Results were assessed after 30 min and 24 h of extended cooling and were expressed as the percentage of viable, moribund and dead sperm.
2.3. Statistical Analysis
Statistical analyses were performed using SAS (SAS Institute Inc., Cary, NC, USA). The effect of treatments on sperm quality parameters was evaluated using a repeated measures mixed model (PROC MIXED) and Tukey's post hoc test. Statistical significance was set at p < 0.05.
3. Results
Table 1 summarises the effect of short‐term cooling (< 1 h), prior to disaccharides supplementation, on sperm motility parameters. There were not significant differences between Fresh and S‐Cooled samples for TM and PM. The kinetics VCL, VAP, VSL and ALH were significantly reduced (p < 0.05) in S‐Cooled.
TABLE 1.
Effect of short‐term cooling (< 1 h) prior to disaccharides supplementation on zebrafish sperm parameters.
| Fresh | S‐Cooled | |
|---|---|---|
| Total motility (%) | 69.86 ± 2.21 | 65.85 ± 3.75 |
| Progressive motility (%) | 63.00 ± 2.25 | 61.17 ± 4.25 |
| VCL (μm/s) | 113.96 ± 4.70 | 83.65 ± 5.95** |
| VAP (μm/s) | 105.46 ± 2.49 | 78.52 ± 6.69* |
| VSL (μm/s) | 95.55 ± 1.83 | 72.95 ± 6.39* |
| STR (%) | 88.60 ± 0.89 | 86.59 ± 1.86 |
| LIN (%) | 81.85 ± 2.36 | 78.50 ± 1.86 |
| WOB (%) | 90.97 ± 1.88 | 88.01 ± 2.46 |
| ALH (μm) | 1.39 ± 0.19 | 0.8 ± 0.02* |
| BCF (Hz) | 14.01 ± 0.31 | 13.72 ± 0.27 |
Note: Asterisks indicate significant differences between Fresh and S‐Cooled; *p < 0.05, **p < 0.01.
Abbreviations: ALH, amplitude of lateral head displacement; BCF, beat cross frequency; LIN, linearity; S‐Cooled, short‐term cooled semen; STR, straightness; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight‐line velocity; WOB, wobble.
Figure 1 shows the effect of sucrose and trehalose addition, after 1 h of short‐term cooling, on TM and PM values obtained during E‐storage for 24 h (1–24 h). Addition of NPC did not improve sperm motility for 60 min of E‐storage. After 24 h, TM and PM were significantly higher in the E‐S13 and E‐T13 treatments than in E‐C and E‐T26. Similar results were obtained for kinetic parameters (Table 2).
FIGURE 1.
Total and progressive motility of extended cooled storage treatments after disaccharide supplementation over time (1–24 h). E‐C = Control, E‐S13 = Sucrose 0.13M + 1% BSA, E‐T13 = Trehalose 0.13M + 1% BSA, E‐T26 = Trehalose 0.26M + 1% BSA.
TABLE 2.
Kinetic parameters of extended cooled storage treatments over time.
| VCL (μm/s) | VAP (μm/s) | VSL (μm/s) | STR (%) | LIN (%) | WOB (%) | ALH (μm) | BCF (Hz) | |
|---|---|---|---|---|---|---|---|---|
| 0 min | ||||||||
| E‐C | 83.12 ± 5.52B | 77.80 ± 6.20B | 72.30 ± 5.84B | 87.64 ± 1.38 | 79.45 ± 2.63 | 88.37 ± 2.09B | 0.81 ± 0.02 | 14.06 ± 0.47 |
| E‐S13 | 95.67 ± 6.58A | 91.60 ± 7.09A | 85.32 ± 6.37A | 88.95 ± 1.02 | 83.29 ± 2.00 | 91.76 ± 1.65A | 0.79 ± 0.02 | 13.84 ± 0.29 |
| E‐T13 | 97.16 ± 5.92A | 92.78 ± 6.38A | 85.76 ± 5.77A | 89.03 ± 1.10 | 83.25 ± 2.15 | 91.97 ± 1.75A | 0.83 ± 0.02 | 14.37 ± 0.19 |
| E‐T26 | 96.80 ± 7.08A | 92.48 ± 7.70A | 85.64 ± 7.02A | 89.40 ± 0.95 | 83.33 ± 2.21 | 91.74 ± 1.92A | 0.80 ± 0.02 | 13.85 ± 0.24 |
| 10 min | ||||||||
| E‐C | 84.92 ± 5.47B | 80.94 ± 5.85B | 74.88 ± 5.46B | 86.17 ± 2.00 | 79.79 ± 2.90AB | 90.06 ± 1.80 bc | 0.77 ± 0.02 | 13.55 ± 0.43 |
| E‐S13 | 95.55 ± 6.82A | 91.98 ± 7.18A | 84.48 ± 6.21A | 88.35 ± 0.93 | 83.33 ± 1.79AB | 92.62 ± 1.64AB | 0.78 ± 0.02 | 13.40 ± 0.30 |
| E‐T13 | 98.74 ± 4.77A | 94.78 ± 5.08A | 87.53 ± 3.89A | 89.96 ± 0.57 | 84.97 ± 0.68A | 93.15 ± 0.97A | 0.83 ± 0.02 | 13.92 ± 0.33 |
| E‐T26 | 84.49 ± 6.44B | 79.72 ± 6.85B | 71.97 ± 6.07B | 85.48 ± 1.50 | 77.77 ± 2.62B | 88.81 ± 2.05C | 0.81 ± 0.02 | 12.97 ± 0.46 |
| 20 min | ||||||||
| E‐C | 86.51 ± 5.69 | 81.89 ± 6.32 | 75.23 ± 5.83 | 84.95 ± 2.24 | 78.40 ± 3.51 | 89.38 ± 2.55 | 0.80 ± 0.02 | 12.99 ± 0.47 |
| E‐S13 | 88.77 ± 7.26 | 84.55 ± 7.69 | 76.53 ± 6.78 | 85.93 ± 1.46 | 79.26 ± 2.79 | 90.30 ± 2.11 | 0.79 ± 0.02 | 12.52 ± 0.54 |
| E‐T13 | 95.99 ± 6.49 | 92.22 ± 6.99 | 83.84 ± 6.20 | 88.46 ± 0.99 | 83.11 ± 1.98 | 92.62 ± 1.88 | 0.79 ± 0.02 | 13.77 ± 0.41 |
| E‐T26 | 86.73 ± 10.43 | 81.33 ± 11.67 | 73.63 ± 10.99 | 84.55 ± 3.89 | 75.57 ± 6.90 | 86.51 ± 5.54 | 0.81 ± 0.03 | 13.12 ± 0.63 |
| 30 min | ||||||||
| E‐C | 88.72 ± 6.93 | 84.64 ± 7.77 | 78.12 ± 7.30 | 86.33 ± 2.33 | 80.18 ± 4.16 | 90.37 ± 3.18 | 0.79 ± 0.03 | 13.29 ± 0.47 |
| E‐S13 | 89.98 ± 6.34 | 85.73 ± 6.62 | 76.94 ± 5.19 | 86.57 ± 1.41 | 80.08 ± 2.10 | 91.45 ± 1.88 | 0.81 ± 0.04 | 12.90 ± 0.58 |
| E‐T13 | 93.99 ± 5.81 | 90.16 ± 6.05 | 81.17 ± 4.87 | 87.42 ± 1.52 | 81.89 ± 1.71 | 92.42 ± 1.39 | 0.79 ± 0.02 | 13.27 ± 0.13 |
| E‐T26 | 85.91 ± 6.34 | 80.88 ± 7.01 | 73.07 ± 6.40 | 84.88 ± 1.83 | 76.80 ± 2.81 | 88.11 ± 2.32 | 0.83 ± 0.03 | 12.70 ± 0.66 |
| 60 min | ||||||||
| E‐C | 83.71 ± 4.17AB | 79.66 ± 4.37AB | 74.24 ± 4.03AB | 87.47 ± 0.81 | 81.40 ± 1.42 | 90.90 ± 1.07 | 0.79 ± 0.02 | 13.39 ± 0.41 |
| E‐S13 | 87.24 ± 4.10AB | 82.89 ± 4.54AB | 75.47 ± 3.54AB | 87.42 ± 0.49 | 80.92 ± 1.04 | 90.90 ± 1.43 | 0.80 ± 0.02 | 13.16 ± 0.26 |
| E‐T13 | 91.85 ± 4.21A | 87.53 ± 4.93A | 78.40 ± 4.36A | 86.33 ± 1.46 | 80.32 ± 2.15 | 91.35 ± 1.74 | 0.82 ± 0.03 | 13.19 ± 0.45 |
| E‐T26 | 79.15 ± 5.44B | 74.12 ± 5.77B | 66.48 ± 4.78B | 85.91 ± 1.32 | 77.44 ± 2.53 | 87.89 ± 2.15 | 0.81 ± 0.01 | 13.15 ± 0.26 |
| 24 h | ||||||||
| E‐C | 68.12 ± 4.84A | 60.74 ± 5.52A | 57.58 ± 5.57A | 86.87 ± 1.94A | 74.21 ± 3.93A | 82.12 ± 3.38A | 0.90 ± 0.02A | 13.93 ± 0.66A |
| E‐S13 | 61.07 ± 3.33AB | 53.10 ± 3.91AB | 49.06 ± 3.77AB | 80.19 ± 1.37B | 66.73 ± 2.56A | 78.60 ± 2.45A | 0.90 ± 0.02A | 11.76 ± 0.18B |
| E‐T13 | 57.97 ± 2.99B | 49.74 ± 2.87B | 45.40 ± 2.79B | 79.04 ± 1.15B | 64.68 ± 1.81A | 76.80 ± 1.64A | 0.90 ± 0.03A | 11.76 ± 0.31B |
| E‐T26 | 35.90 ± 2.04C | 25.57 ± 1.84C | 21.15 ± 1.64C | 70.52 ± 1.76C | 46.79 ± 3.71B | 61.83 ± 3.74B | 0.77 ± 0.02B | 8.24 ± 0.61C |
Note: E‐C = Control, E‐S13 = Sucrose 0.13M + 1% BSA, E‐T13 = Trehalose 0.13M + 1% BSA, E‐T26 = Trehalose 0.26M + 1% BSA. Different superscripts (A‐C) indicate significant differences (p < 0.05) among treatments.
Figure 2 shows PMI after 30 min and 24 h of E‐storage. After 30 min (Figure 2A), the percentage of dead spermatozoa was significantly higher in E‐T26. After 24 h (Figure 2B), the proportion of viable spermatozoa was also lower in E‐T26.
FIGURE 2.
Comparison of sperm membrane integrity at cooling storage times 30 min and 24 h. E‐C = Control, E‐S13 = Sucrose 0.13M + 1% BSA, E‐T13 = Trehalose 0.13M + 1% BSA, E‐T26 = Trehalose 0.26M + 1% BSA.
4. Discussion
The evaluation of the effect of short‐time cooling (< 1 h) on sperm motility agreed with the findings of Cheng et al. (2021). Although there were no significant differences on TM and PM in Fresh and S‐Cooled, several kinetic parameters decreased by 1 h of cool storage, indicating that even short storage may affect sperm velocity and flagellar activity (Contreras et al. 2020).
These findings highlight the importance of optimising cooling extenders in zebrafish semen. Our results showed that the addition of sucrose or trehalose at 0.13 M obtained higher values of motility parameters after 24 h of cold storage. However, higher concentrations of trehalose (0.26 M) decreased sperm motility. Flow cytometry analysis also revealed important effects of NPCs on PMI. E‐C sperm showed similar PMI values to E‐S13 and E‐T13 after 30 min and 24 h of storage, consistent with previous reports that have described possible tolerance of fish sperm to cold shock (Contreras et al. 2020). E‐T26 yielded a higher proportion of dead spermatozoa than E‐C, suggesting that this high concentration destabilises the sperm membrane and decreases sperm motility (Yang et al. 2016).
These results seem to agree with those reported in previous studies in which the effect of sugars was studied for sperm preservation (de Almeida‐Monteiro et al. 2025; Anjos et al. 2021; Zalazar et al. 2024; Zhu et al. 2016). According to these studies, disaccharides exert their protective effect by increasing the osmotic pressure in the extracellular medium. During the initial equilibration period, this change could cause osmotic stress that may affect sperm parameters, particularly at high disaccharide concentration (0.26M). After longer storage periods (> 24 h), supplementation with sucrose or trehalose 0.13M may protect sperm from spontaneous movement caused by osmolarities lower than 320 mOsm/kg (Cheng et al. 2021). These results highlight the importance of media osmolarity for zebrafish preservation.
In conclusion, the addition of sucrose and trehalose 0.13M improved sperm motility after 24 h of storage at 5°C. Further studies are needed to target the exact time at which disaccharide addition begins exerting a beneficial effect on zebrafish sperm.
Author Contributions
B.R.N.V.: conceptualisation, methodology, investigation, data curation, writing – original draft. P.V.‐S.: investigation, writing – review and editing. C.M.‐P.: investigation, writing – review and editing. M.H.: resources, funding acquisition. N.A.: resources. R.M.: resources. L.D.S.M.: methodology, writing – review and editing. J.D.: resources, writing – review and editing, funding acquisition. I.O.: conceptualisation, methodology, formal analysis, writing – review and editing.
Funding
This study was supported by the project grant PID2020‐116090RB‐I00 from MICIN (Ministerio de Ciencia e Innovación), which also supported CMP. PVS was supported by the Spanish FPU fellowship (FPU23/03472) from Ministerio de Universidades. BRNV was funded in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001.Funding for open access charge: Universidad de Cordoba/CBUA.
Conflicts of Interest
The authors declare no conflicts of interest.
Acknowledgements
Special thanks to the personnel of the Servicio de Animales de Experimentación (SAEX) of Universidad de Córdoba.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.