Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Abstract

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

A Simple and Efficient Semen Cryopreservation Method to Increase the Genetic Variability of Endangered Mediterranean Brown Trout Inhabiting Molise Rivers

“…Other authors also found that a higher thawing temperature ensured better sperm cryosurvival [9,13,15,16]. On the contrary, in our previous research, the thawing temperature of 10 • C registered both a higher post-thaw semen quality and fertilization ability with respect to those recorded at 30 • C/10 s [7]. These conflicting results could be attributed to the different experimental conditions adopted in the two studies (extender composition, freezing rates and equilibration time).…”

“…The freezing procedure tested here turned out to have a positive impact on sperm total motility post-thawing, recording values above 80%. These results were remarkably better compared to that found in our previous studies, which ranged between 33% and 52% [5][6][7]. However, a significant decrease in the total motility after thawing compared to the fresh semen was recorded, although this is expected following the cryopreservation process.…”

“…Although, we did not obtain the same excellent results that were reported our Polish colleagues who recorded a fertilization rate close to that of fresh semen, we think that the freezing semen protocol standardized here resulted as effective even for the autochthonous trout populations from Molise. It is a practical freezing protocol that includes the use of a simple glucose-methanol extender and a reduced sperm-to-egg ratio by a 10 factor, resulting as better with respect to our previous freezing protocol [7] whilst maintaining similar fertilization rates (5 × 10 6 :1 sperm/egg ratio; 54.5% fertilization rate vs. 4.5 × 10 5 :1 sperm/egg ratio; 53.2% fertilization rate).…”

“…They were then placed in liquid nitrogen. The straws were then thawed by immersion in a water bath at two different thawing rates: at 40 • C for 5 s, as reported in the original protocol by Nynca et al [10], or at 10 • C for 30 s, as already used in our previous work [7].…”

“…Thus, the conservation of cell structure and its functionality strictly depends on the cryopreservation protocol used. In this regard, in the past years our research group has been perfecting an effective semen cryopreservation protocol for the Mediterranean trout of the Molise rivers, with the aim of reducing cell damage and preserving the fertilizing ability of sperm [5][6][7]. However, despite our efforts and promising results obtained so far, we still aim to obtain a freezing procedure reporting a fertilization rate as similar as possible to that of fresh semen.…”