Fertilizing Capacity of the Cryopreserved Sperm of Prochilodus brevis

Abstract

Background: Seminal cryopreservation is a technique that optimizes aquacultural production, as it requires less breeding and enables reproduction outside of the breeding season. This technique also helps to preserve species, thus reducing the pressure on the natural stocks. Several studies have sought to develop freezing protocols that result in semen of a good quality. However, some studies do not evaluate the ability of frozen semen to produce viable larvae. Therefore, the aim of this study was to verify the fertilizing capacity of the frozen semen of Prochilodus brevis.

Materials, Methods & Results: Semen from twenty adult males of the Brazilian bocachico was collected and evaluated to establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, membrane integrity, pH, and concentration. Six pools were formed, each of which was diluted in a freezing medium containing 5% glucose with 10% dimethyl sulfoxide (DMSO) or 5% glucose with 10% methyl glycol (MG). The samples were loaded into 0.25 mL French straws, frozen in a dry shipper, and stored in a liquid nitrogen canister. The semen was then thawed and evaluated to establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, and membrane integrity. For the fertilization test, four females were used. The oocytes from each female were divided into three batches and fertilized with either fresh or cryopreserved semen. The rates of fertilization, hatching, and larval survival were then measured. Data were expressed as the mean ± standard deviation and analyzed using SAS (2002). The frozen semen with glucose + DMSO was significantly higher (P < 0.001) than the frozen semen with glucose + MG, in all seminal quality parameters evaluated (63.95 ± 15.88% and 25.36 ± 3.53% for the motility, 36.38 ± 7.02 μm.s^-1 and 20.45 ± 2.84 μm.s^-1 for the curvilinear velocity, 19.26 ± 2.74 μm.s^-1 and 3.03 ± 1.40 μm.s^-1 for the straight linear velocity, 25.70 ± 6.51 μm.s^-1 and 6.90 ± 1.12 μm.s^-1 for the average path velocity, and 63.58 ± 6.95% and 35.58 ± 11.26% for the membrane integrity, respectively). MG were very close to zero and not statistically significant. Regarding these same parameters, there were no significant differences (P > 0.05) when the fresh semen was compared to the cryopreserved semen with glucose + DMSO (36.25 ± 2.5% and 29.16 ± 5.64% for the fertilization rate, 38.56 ± 11.23% and 29.33 ± 11.75% for the hatching rate, and 11.59 ± 5.16% and 7.63 ± 5.46% for the larval survival rate, respectively).

Discussion: This is the first study of the artificial fertilization of Prochilodus brevis using cryopreserved semen. Seminal quality parameters are important for predicting the success of the cryopreservation technique, however, in vivo tests are essential to confirm such success. Thus, obtaining larvae is a major step towards the standardization of a cryopreservation protocol for a particular species. It is known that cryopreservation reduces the seminal quality but is a necessary process for the conservation of male gametes in the long term and, as shown in this study, good results can be obtained. In this study, the best results were obtained with the inclusion of DMSO in the freezing medium. This effect can be attributed to DMSO having a very low molecular weight, which decreases the formation of ice crystals. Considering the results obtained, we concluded that it is feasible to obtain larvae of the Brazilian bocachico using frozen semen in a 5% glucose solution with 10% DMSO.