Short communicationMotility activation of rainbow trout spermatozoa at pH 6.5 is directly related to contamination of milt with urine
Introduction
Contamination of milt with urine is of major importance regarding the sperm quality of freshwater fish. Such contamination is unavoidable under the conditions of milt collection by stripping, because the spermatic duct and urinary duct join together into the efferent duct with an opening through the urogenital papilla. The urine of teleost fish is of low osmolality therefore the mechanism of its detrimental effect is explained by the induction of partial activation of sperm motility, as a matter of fact spontaneous movement of spermatozoa in collected semen is observed (Linhart et al., 2003). Initiation of sperm movement is accompanied by a reduction of intracellular ATP store (Poupard et al., 1998). Other cellular events related to urine contamination are unknown at present.
In our recent study we studied the pH-dependence of sperm motility activation in several Salmoniformes species (Ciereszko et al., 2010). Species-specific variability was observed, however, at least partially, this variability could indirectly be linked to the urine contamination of milt. The most striking difference in the sperm motility activation pattern, was found between milt samples collected from rainbow trout (Oncorhynchus mykiss) by stripping and by use of a catheter. In the latter , motility was observed at pH 7.0 and below, contrary to milt collected by stripping when no motility was recorded in that pH range. We suggested, that lack of motility of stripped milt at lower pH values is caused by the detrimental effect of urine introduced into milt during stripping. In this study, we aimed to provide direct evidence to support this suggestion. We collected urine and milt separately using a catheter and then experimentally introduced urine to milt at 10–30%. Sperm motility at pH 6.5 was measured to evaluate the effects of contamination. We selected a pH of 6.5, because at this pH samples collected by stripping are usually immotile (Ciereszko et al., 2010). At the same time, sperm motility in D532 solution (which is optimal for rainbow trout motility measurement and fertilization, Billard, 1992) was measured in order to monitor changes in sperm motility at optimal conditions for motility activation. By using this approach we could evaluate if the lack of motility at pH 6.5 is related to the permanent inability of spermatozoa to be activated. In other words, we could gauge if sperm, immotile at pH 6.5, can still be activated under optimal conditions.
Section snippets
Milt and urine collection
Milt was collected with a catheter from 3-year old rainbow trout (n = 5) maintained in the Rutki Salmonid Research Laboratory, Institute of Inland Fisheries, Poland. Urine was collected from three male rainbow trout using a catheter and pooled prior to the experiment. Approval by the Animal Experiments Committee in Olsztyn, Poland was gained before starting any of the experiments.
Effects of urine contamination on sperm motility
Semen samples from each individual were mixed with 10, 20 and 30% of urine. The final volume of suspensions was 200 μl.
Effects of the addition of urine to milt on seminal plasma osmolality
The sperm concentration of milt samples was established to be 9.9 ± 2.2 × 109 spermatozoa x ml− 1 and osmolality of pooled urine sample was 25 mOsm/kg. Dose-dependent decline in osmolality of seminal plasma was recorded for semen samples contaminated with urine. Contamination of rainbow trout milt with urine led to dose-dependent decrease in seminal plasma osmolality, 301 ± 3, 261 ± 4, 235 ± 4, and 203 ± 2 mOsm/kg, for 0, 10, 20 and 30% urine contamination, respectively. The equation of linear regression
Discussion
In this study, we have demonstrated that experimental contamination of rainbow trout milt with urine can lead to significant dose-dependent decrease in seminal plasma osmolality. Such contamination significantly reduced the ability of rainbow trout spermatozoa to be activated at pH 6.5. On the other hand, no changes of motility in D532 (pH 9.0) were observed at the same time.
High variability of seminal plasma osmolality of rainbow trout has been demonstrated in several studies and exceptionally
Acknowledgements
This study was supported by funds appropriated to Institute of Animal Reproduction and Food Research.
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