Cryopreservation of coho salmon sperm (Oncorhynchus kisutch): Effect on sperm function, oxidative stress and fertilizing capacity

Abstract

Sperm cryopreservation is an important tool for reproductive management; however, it has not been possible to perform this procedure for coho salmon. This study was designed to assess the effect of cryopreservation on sperm functional parameters, oxidative stress markers and fertilizing capacity of coho salmon. Eight extenders were tested, consisting of two intracellular cryoprotectant agents (CPA-I): dimethylsulfoxide (DMSO) at 8 and 10% and methanol (MeOH) at 9 and 12%, two extracellular cryoprotectants (CPA-E): 0.14 M trehalose and 0.18 M glucose, supplemented in all cases with 10% egg yolk and Storfish®. Milt samples from 18 males were evaluated in three trials. In the first trial, the toxic effect of cryoprotectants on motility was evaluated after 0, 15, 30, and 60 min of equilibration time. The second trial was conducted to select the extender which produced the best post-thaw sperm motility. In both trials, sperm motility was analysed by a subjective method. In the third trial, milt samples were frozen in the three best extenders; post-thawed samples were assessed for motility by computer-assisted sperm analysis CASA, viability, superoxide anion production (O⁠2⁠•−), mitochondrial membrane potential (ΔΨm) by flow cytometry, lipid peroxidation (malondialdehyde, MDA) and fertilizing capacity were also analysed. In Trial 1, no negative effect of the extenders tested was observed on sperm motility during the different equilibration times. In Trial 2, the post-thaw sperm motility was significantly affected by CPA-I, CPA-E and their interaction, in other words, the protection capacity was dependent on the type of CPA-I and at the same time on the CPA-E. Thus, the best results were obtained with DMSO combined with glucose, with mean motility of 16.44 ± 12.55% (8% DMSO + glucose) and 11.57 ± 10.23% (10% DMSO + glucose). In Trial 3, all the sperm parameters were significantly affected after freezing. Mean post-thaw sperm motility varied from 20.50 ± 15.83% to 35.80 ± 17.64%, cell viability ranged from 56.23 ± 11.05% to 59.42 ± 12.00% and the ΔΨm varied from 12.50 ± 8.9% to 14.63 ± 10.09%, without significant differences between the three extenders assessed in this trial. On other hand, the percentage of living spermatozoa producing O⁠2⁠•− and the MDA levels increased significantly after freezing, which suggests that sperm cryodamage was caused in part by oxidative stress. The combination of either 8% or 10% DMSO with glucose yielded the best fertility rates (30.00 ± 11.73% and 31.00 ± 10.25% respectively). In conclusion, DMSO in combination with glucose and hen egg yolk is the most suitable extender for preserving the sperm quality and fertilizing capacity of coho salmon.