Abstract
Simple SummaryCryobanking is an important tool to preserve the genetic resources of fish species. Semen cryopreservation has been extensively used in conservation programs for endangered species. Here, we aimed to find an effective cryopreservation protocol for the autochthonous Mediterranean brown trout inhabiting the Biferno river (south Italy), in order to create a sperm cryobank. Low-density lipoproteins and sucrose were tested as non-permeating cryoprotectants (NP-CPAs) to replace the egg yolk. Moreover, the thawing rate (10 °C for 30 s vs. 30 °C for 10 s) was also studied. From results obtained in vitro and in vivo, egg yolk emerged as the best NP-CPA and the lower thawing rate recorded better post-thaw semen quality in vitro and higher fertilization and hatching rates in vivo. These findings are important because they will contribute to the creation of a sperm cryobank for Molise’s native trout, which is a milestone of our European project (Life Nat.Sal.Mo).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.
Highlights
Sperm cryopreservation is considered a valuable tool for preserving the genetic material of endangered fish species by storage of their gametes in a cryobank [1,2,3]
The data obtained indicated a significant effect for the concentrations of non-permeating cryoprotectants (NP-CPAs) and thawing rates on all parameters considered, except for on the motility duration for the thawing rate effect
The results clearly demonstrated that the type and concentrations of NP-CPAs used affect the post-thaw quality of trout semen
Summary
Sperm cryopreservation is considered a valuable tool for preserving the genetic material of endangered fish species by storage of their gametes in a cryobank [1,2,3]. The semen cryobanks provide the opportunity to preserve representative samples and further reconstruct the original strain, population, or diversity [2,4] In this regard, we attempted to find an effective semen cryopreservation protocol for the Mediterranean brown trout (Salmo cettii), a native species that inhabits the rivers of Molise [5]. We attempted to find an effective semen cryopreservation protocol for the Mediterranean brown trout (Salmo cettii), a native species that inhabits the rivers of Molise [5] This species is listed in the Italian IUNC Red List as critically endangered [6], due to river pollution, uncontrolled fishing, and hybridization following the introduction of non-native strains that have drastically reduced the number of the native species in recent centuries [7]. An adequate amount of satisfactory results had been obtained when the semen was frozen in the presence of a glucose-DMSO extender at 5 cm above the liquid nitrogen surface, for 10 min
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