Introduction
Nowadays, the most used fish sperm cryo-containers are straws made of polypropylene or polyvinyl chloride (PVC). They cannot be reused, making them waste with a high potential for polluting the environment. Thus, we aimed to assess the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws for fish sperm cryopreservation.
Material and Methods
South American silver catfish Rhamdia quelen sperm samples (n = 12) were cryopreserved in 5% D-fructose + 5% powdered milk + 10% methanol diluted in a ratio 1:4 (sperm: cryo-solution). European eel Anguilla anguilla sperm samples (n = 12) were cryopreserved using P1 extender + 5% egg yolk + 10% methanol diluted in a ratio 1:9 (sperm: cryo-solution). Gilthead seabream Sparus aurata sperm samples (n = 12) were cryopreserved in NAM (non-activating medium) extender with 10% dimethyl sulfoxide (DMSO) after dilution in a ratio 1:6 (sperm: cryo-solution). European sea bass sperm samples (n = 10) were diluted in NAM at 1:5.7 (sperm: NAM), and 5 % DMSO was added. Sperm samples of all species, with motility over 60%, were individually cryopreserved in plastic straws, hard-gelatin, and hard-hydroxypropyl methylcellulose (HPMC) biodegradable capsules. All the containers were filled (silver catfish and seabream – 0.25 mL; eel and sea bass – 0.5 mL) and cryopreserved in liquid nitrogen vapor. The straws were thawed in a water bath (silver catfish 25 °C/10 s, eel 40 °C/13 s, seabream 60 °C/5 s, and sea bass 35 °C/ 15 s), and the capsules were thawed and dissolved into a plastic tube (15 mL) containing a previously warmed extender (5 mL- silver catfish 25 °C, eel and seabream 40 °C, and sea bass 35 °C) under shake in a vortex for 25 s.
Total motility (MOT - %), curvilinear (VCL - µm/s), straight line (VSL - µm/s), and average path (VAP - µm/s) velocities were evaluated by CASA-Mot software. Rates of fertilization, hatching, and normal larvae morphology were determined using silver catfish post-thawed sperm. The alkaline comet assay was performed to quantify sperm DNA damage of eel, gilthead seabream, and sea bass. Tail-DNA (%) was evaluated using CaspLab software.
Results
In all four species, sperm cryopreservation led to a MOT decrease in all the storage containers (Fig. 1). Nonetheless, no significant differences in MOT were found between samples preserved in capsules and straws. A similar result was observed in sperm velocity parameters. Eel sperm cryopreserved in straws retained velocity values comparable to fresh samples, while those stored in gelatin capsules preserved their VSL and VAP post-cryopreservation. For sea bass, sperm stored in gelatin capsules exhibited higher velocities than those preserved in HPMC capsules.
There was no difference between straw and capsules in silver catfish fertilization, hatching, and normal larvae rates. Cryopreservation did not result in detectable DNA fragmentation in eel and sea bass sperm. Conversely, Tail DNA of gilthead seabream sperm cryopreserved in gelatin (10 ± 2%) and HPMC (11 ± 6%) capsules increased after thawing when compared to fresh (4 ± 3%) and straw-stored (4 ± 1%) samples.
Discussion
During cryopreservation, cells are exposed to high thermal and osmotic stress, mainly during freezing and thawing, causing damage to the cells (Mazur et al., 1972) and impairing sperm motility of all four species. However, the kinetic parameters of sperm samples cryopreserved in straws and capsules did not differ. It was noted that silver catfish samples cryopreserved using all three container types achieved fertilization rates of approximately 70%, a value considered high according to Beirão et al. (2019). We did not observe increased sperm DNA damage in European eel and sea bass after freezing and thawing. However, we observed a Tail DNA of 10 and 11% in sperm cryopreserved in gelatin and HPMC capsules, respectively. These results indicate low sperm DNA damage, and the oocyte could probably repair this level of damage during early embryogenesis.
Based on our results, hard-gelatin and HPMC biodegradable capsules can be an alternative to plastic straws for South American silver catfish, European eel, gilthead seabream, and European sea bass sperm cryopreservation.
Acknowledgments
This study forms part of the ThinkInAzul programme and was supported by MICIU with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana [GVA-THINKINAZUL/2021/012, -24, -42; Principal investigators: J.F. Asturiano (Universitat Politècnica de València), J. Pérez-Sánchez, and A. Gómez (CSIC), respectively]. FAPERGS (17/2551-0000917-7), CAPES/PROEX (8882.346090/2015-1), CNPq support to Thales França (141717/2019-0 and 200285/2021-1).
References
Beirão, J., Boulais, M., Gallego, V., O’Brien, J. K., Peixoto, S., Robeck, T. R., Cabrita, E. (2019). Sperm handling in aquatic animals for artificial reproduction. Theriogenology, 133, 161–178.
Mazur, P., Leibo, S. P., Chu, E. H. Y. (1972). A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp. Cell. Res., 71, 345–355.