W. Demianowicz

Fertilization rate of Siberian sturgeon (Acipenser baeri, Brandt) milt cryopreserved with methanol

Milt obtained from three Siberian sturgeon males (Acipenser baeri, Brandt) were cryopreserved using three extenders: Tris–sucrose–KCl (30 mM Tris, 23.4 mM sucrose, 0.25 mM KCl, pH 8.0), Tris–NaCl (10 mM Tris, 25 mM NaCl, pH 8.5), and Tris–sucrose (20 mM Tris, 400 mM sucrose, pH 8.0) supplemented with 10% methanol. Semen was diluted 1:1 with appropriate extender and frozen in liquid nitrogen vapor. After fertilization with cryopreserved milt, hatching rates of 29.6±5.0%, 18.2±2.4%, and 6.0±3.0% were recorded for Tris–sucrose–KCl, Tris–NaCl, and Tris–sucrose extender, respectively. Rates for the first two extenders were similar to data of fresh semen obtained from two males(17.9% and 26.0% for male #1 and #2, respectively). Our results indicate that Tris–sucrose–KCl and Tris–NaCl are useful extenders and methanol is a useful cryoprotectant for cryopreservation of sturgeon semen.

Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success

In the current work, seminal plasma was used for the first time as an incubation medium for monitoring short-time exposure effects of sublethal concentrations of mercury and cadmium ions on rainbow trout sperm. Sperm motility parameters (CASA) and hatching rates were used as gamete quality markers. Additionally live/dead sperm viability test and comet assay of DNA fragmentation were performed. We demonstrated that computer-assisted sperm motility analysis (CASA) may serve as a predictor of reproductive success, when milt contaminated with heavy metals is used. Results presented in this study demonstrate that mercury ions altered sperm motility characteristics at 1-10 mg Hg2+/l and 10 mg Cd2+/l and hatching rates at 10 mg Hg2+/l and 10 mg Cd2+/l after 4h of exposure. Although mercury ions affected sperm motility parameters immediately after dilution with milt as well as at 4h of exposure, no differences in sperm motility parameters were found between intact and mercury-treated milt after 24h of exposure. Our results suggest that rainbow trout seminal plasma has a protective role against the toxic effects of mercury ions of rainbow trout sperm motility.

Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa

The effects of extender composition and equilibration time on fertilizing ability of cryopreserved spermatozoa from rainbow trout, Oncorhynchus mykiss, were investigated. In addition, enzyme activity in supernatants from thawed sperm was assessed. The use of the two extenders: Erdahl & Grahams + 10% DMA (dimethyl acetamide) + 10% egg yolk and 0.3 M glucose + 10% DMA yielded the highest post-thaw fertilization rates. We observed interactions between extender constituents and the equilibration of diluted semen. This indicates a multifactorial effect of the extender constituents on spermatozoal resistance against injuries. The 10-min equilibration of spermatozoa in extender before freezing generally lowered the fertilization ability of spermatozoa, except for DMA-based extenders. The addition of egg yolk to the extender was generally beneficial, especially in DMA- and DMSO-based extenders. The use of low-density lipoprotein fraction showed no advantage to full-yolk or free-of-yolk extenders. Aspartate aminotransferase and lactate dehydrogenase leakage from damaged spermatozoa correlated negatively with the ability of cryopreserved spermatozoa to fertilize eggs. Each factor tested, when analyzed separately, did not give general information about its effect on the fertilization ability of cryopreserved sperm. The multifactorial analysis of the important factors in cryopreservation of trout spermatozoa showed their cumulative effect. This is the most likely reason for divergent information reported elsewhere on the effect of various factors in the cryopreservation of rainbow trout spermatozoa.

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.