Sperm cryopreservation is useful for the conservation of endangered fish species. This procedure has effectively been described as safe for the ex situ preservation of biodiversity by facilitating the storage of gametes in a gene bank. The success of fish semen cryopreservation depends on interplay among many factors (freezing medium, cryoprotectant (CPA) and its concentration, equilibration time, and freezing and thawing rate). The choice of equilibration time is certainly among the most important factors for an effective semen freezing protocol. The equilibration time is the period of time necessary for the CPA to permeate the sperm. Accordingly, the objective of this study was to determine the effects of two equilibration times on post-thaw semen quality of Mediterranean brown trout from the Biferno river. Twenty autochthonous Mediterranean brown trout males (Salmo cettii) were captured from the Biferno river (Molise region) by electro-fishing. Semen was collected by gentle abdominal massage. Ejaculates were pooled (4 ejaculates/pool), 6 pools were used. Each pool was divided into two subsamples and diluted 1:3 in freezing extender containing 300 mM glucose, 10% egg yolk and 10% dimethylsulfoxide. The extended semen was loaded in 0.25 mL plastic straws and equilibrated at 4 C for 10 or 30 min (equilibration time). At the end of each period, the semen was frozen by exposure to liquid nitrogen vapor (5 cm above the liquid nitrogen surface) for 10 min; then, the straws were plunged into liquid nitrogen (-196 C). Semen samples were thawed at 30 C for 10 s. Sperm motility, spermatozoa movement duration (SMD), sperm viability (SyBr-PI) and sperm DNA integrity (Acridine orange) were assessed in both fresh and frozen semen. Semen quality parameters recorded in fresh semen show a good quality soon after collection. After thawing, no significant effect of the equilibration time (10 vs 30 min) was observed for all the sperm quality parameters assessed. However, slightly higher values were observed with 10 min equilibration compared to 30 min: sperm motility ¼ 33.5% vs 31.8%, SMD¼ 40.7% vs 38.7%, viability¼ 36.3% vs 35.9%. These results lead us to assume that the equilibration time of 10 minutes is sufficient to allow dehydration of sperm cells. The development of an effective freezing protocol is required to create a sperm cryobank to support the ex situ in vitro conservation of the original population of Mediterranean brown trout in the Biferno river.
Effect of equilibration time for semen cryopreservation of endangered Mediterranean brown trout (Salmo cettii) inhabiting the Biferno river (Molise region, South Italy)
IAFFALDANO N.;DI IORIO M.;
2013-01-01
Abstract
Sperm cryopreservation is useful for the conservation of endangered fish species. This procedure has effectively been described as safe for the ex situ preservation of biodiversity by facilitating the storage of gametes in a gene bank. The success of fish semen cryopreservation depends on interplay among many factors (freezing medium, cryoprotectant (CPA) and its concentration, equilibration time, and freezing and thawing rate). The choice of equilibration time is certainly among the most important factors for an effective semen freezing protocol. The equilibration time is the period of time necessary for the CPA to permeate the sperm. Accordingly, the objective of this study was to determine the effects of two equilibration times on post-thaw semen quality of Mediterranean brown trout from the Biferno river. Twenty autochthonous Mediterranean brown trout males (Salmo cettii) were captured from the Biferno river (Molise region) by electro-fishing. Semen was collected by gentle abdominal massage. Ejaculates were pooled (4 ejaculates/pool), 6 pools were used. Each pool was divided into two subsamples and diluted 1:3 in freezing extender containing 300 mM glucose, 10% egg yolk and 10% dimethylsulfoxide. The extended semen was loaded in 0.25 mL plastic straws and equilibrated at 4 C for 10 or 30 min (equilibration time). At the end of each period, the semen was frozen by exposure to liquid nitrogen vapor (5 cm above the liquid nitrogen surface) for 10 min; then, the straws were plunged into liquid nitrogen (-196 C). Semen samples were thawed at 30 C for 10 s. Sperm motility, spermatozoa movement duration (SMD), sperm viability (SyBr-PI) and sperm DNA integrity (Acridine orange) were assessed in both fresh and frozen semen. Semen quality parameters recorded in fresh semen show a good quality soon after collection. After thawing, no significant effect of the equilibration time (10 vs 30 min) was observed for all the sperm quality parameters assessed. However, slightly higher values were observed with 10 min equilibration compared to 30 min: sperm motility ¼ 33.5% vs 31.8%, SMD¼ 40.7% vs 38.7%, viability¼ 36.3% vs 35.9%. These results lead us to assume that the equilibration time of 10 minutes is sufficient to allow dehydration of sperm cells. The development of an effective freezing protocol is required to create a sperm cryobank to support the ex situ in vitro conservation of the original population of Mediterranean brown trout in the Biferno river.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
