Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes
Thales S. França, Wendy A. González-López, Malbelys P. Sanchez, Leonor Ferrão, Fátima Fernández-García, Laís P. Borges, Álvaro Belenguer, Paul G. Holhorea, Josep C. Calduch-Giner, Alicia Felip, Ana Gómez, Jaime Pérez-Sánchez, Danilo P. Streit Jr, Juan F. Asturiano
Abstract
We aimed to evaluate the efficiency of hard-gelatin
and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable
alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European
sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples
from each European eel (n=12) were diluted 1:8:1
(sperm: extender P1+5% egg yolk: methanol). Gilthead seabream (n=12) samples
were individually diluted in a cryoprotectant solution of 5% Me2SO +
NaCl 1% plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm:
cryoprotectant solution). European sea bass (n=10) sperm from each male was
diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5%
of Me2SO was added. The diluted European eel and sea bass sperm
aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL),
hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm
(0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules
described. All samples were frozen in liquid nitrogen vapor and stored in a
liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot
software. Sperm membrane integrity was performed using a Live and Dead KIT and
an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay
was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by
CaspLab software. Sperm cryopreservation of the three Mediterranean species in
straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity.
Generally, the post-thawing samples cryopreserved in straws and capsules did
not differ for the kinetic parameters and cell membrane integrity, except for
European sea bass sperm, where the samples stored in gelatin capsules showed
higher velocities (VCL - 100; VSL - 76; VAP - 90 µm/s)
than the sperm stored in HPMC capsules (VCL - 87; VSL -
59; VAP - 73 µm/s). The cryopreservation process did not damage the sperm
DNA of European eel and European sea bass, regardless of the containers used.
On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8%; OTM - 9.7) and HPMC (TD - 11.1%; OTM -
11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6%; OTM - 2.7) and the sperm stored in straws (TD - 4.4%; OTM - 5.2). The hard-gelatin and HPMC
biodegradable capsules can be used as an alternative to straws for European
eel, gilthead seabream, and European sea bass sperm cryopreservation.
Available here
.jpg)
