The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

Abstract

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout (Oncorhynchus mykks) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol/L (5 %) dimethylsulfoxide (DMSO) and 0.13 mol/L (1 %) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5 %,I-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15 %])andpropanediol(l.24mol/L[10%]-1.78 mol/L[15 "/o]). Addition of hen egg yolk (7 %, 20 Yo) and of 15 mmol/L (0.5 %)sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5 YO, 3 YO) had no influence on the investigated semen parameters. lntroductionCryopreservation of fish semen is still a field of intensive research (for review see Jamieson 1991). Numerous studies have been performed especially in salmonid fishes (e.Piironen 1993). However, the techniques vary greatly (e.g. extender composition and cryoprotectants, freezing and thawing conditions, fertilization technique) (Scott and Baynes 1980;Stoss 1983;Billard 1992) and no generally accepted concept has yet been found. Evaluation of physiological and biochemical semen parameters could give more information about alterations and damages of spermatozoa during cryopreservation (Malejac et al. 1990;Billard 1992), but most studies are based on fertilization assays only.Therefore we investigated the influence of some commonly used cryoprotectants on motility parameters (computer-assisted cell motion analysis), eosin permeability (sperm cells viability test) and enzyme (lactate dehydrogenase) activities which were released from sperm cells of the rainbow trout (Oncorhynchus mykiss). Since no good, theoretical model is available for the freezing of salmonid semen, a conventional buffered physiological saline solution and freezing in straws are used as basis. Materials and methods Collection of gametesMature male rainbow trout (Oncorhynchus mykiss, winter strain, age 2-4 years) were obtained from the Kreuzstein (Salzburg) fish farm. Fishes used for the experiments were in a running stage and in the middle of the spawning season. The urinary bladder was cleared by gentle abdominal pressure and thereafter the semen was collected by stripping, giving 5-8 ml per male. Sperm density was 8-10 x lo9 spermatozoa x ml-' (determined in a Burker Turk counting chamber, 1000-fold dilution).Samples contaminated with blood, urine and faeces were discarded. T...

Exploration of natural cryoprotectants for cryopreservation of African catfish, Clarias gariepinus, Burchell 1822 (Pisces: Clariidae) spermatozoa

“…In addition, egg yolk has been tested as a cryoprotectant in many cryopreservation studies, for example in yellow perch (Ciereszko et al 1993) and rainbow trout (Lahnsteiner et al 1996). However, these potential cryoprotectants have never been tested for African catfish spermatozoa.…”

Protein concentration and enzyme activities of fresh and frozen–thawed Persian sturgeon, Acipenser persicus (Borodin, 1897) semen

“…For this reason, they are good potential candidates as markers of cryoinjury, as was previously described for teleost fish (Glogowski et al 1996;Lahnsteiner et al 1996;Babiak et al 1997). Lactate Dehydrogenase and AcP activities may be indicators of damage to the plasmolemma and the midpiece area of the sperm structure, whereas AS and b-NAGase activities may be useful indices of damage to the acrosome in cryopreserved semen.…”

Inducción hormonal de la espermiación y criopreservación de esperma en anguila europea (Anguilla anguilla).

“…El glicerol se utiliza en ciertas especies de salmónidos (Stein y Bayrle, 1978), la DMA (dimetilacetamida) se utiliza en esperma de trucha arcoiris (Oncorhynchus mykiss) (Nagoya et al 1996); mientras que combinaciones de DMSO con glicerol dieron buenos resultados en trucha arcoiris (Lahnsteiner et al 1996).…”

“…Aunque, por otro lado, las enzimas pueden provocar la desnaturalización de las proteínas a altas temperaturas y provocar toxicidad en los sistemas celulares (Chao, 1991). Existe la hipótesis de que determinadas sustancias podrían interactuar con la membrana plasmática manteniendo la estabilidad estructural durante el proceso de congelación y descongelación (Anchordoguy et al 1988;Lahnsteiner 1996;He y Woods, 2003). Estudios previos de Ohta (2000, comunicación personal) en los que se empleaban medios con FBS ya hacían referencia a buenos resultados en cuanto a incrementos en la movilidad espermática post-congelación en la anguila japonesa.…”

“…Las primeras investigaciones se iniciaron en semen de bóvidos y posteriormente han abarcado diversas especies de mamíferos tales como ratones, conejos y especialmente humanos (Chereguini et al 1992). En especies de vida acuática, son los peces los que han sido ampliamente estudiados, más aún, los primeros éxitos de fecundación de huevos con espermatozoides criopreservados se realizaron en especies de agua dulce, especialmente en salmónidos, tilapia y carpa (Chereguini et al 1992;Lahnsteiner et al 1996). Estos estudios se han extendido a la criopreservación de esperma de numerosas especies diferentes de peces de agua dulce y marina.…”