Aquaculture 300333 (2012) 185188

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Aquaculture
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Short communication

Motility activation of rainbow trout spermatozoa at pH 6.5 is directly related to contamination of milt with urine
J. Nynca a, G.J. Dietrich a, H. Kumiski b, S. Dobosz b, A. Ciereszko a,
a b

Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Science, 10-748 Olsztyn, Poland Department of Salmonid Fish Research, Inland Fisheries Institute, Rutki, 83300 ukowo, Poland

a r t i c l e

i n f o

a b s t r a c t
Semen samples (collected using a catheter, n = 5) were experimentally contaminated with urine (10 30%) and incubated for 2 and 4 h. The percentage of sperm motility was measured at pH 6.5 and in D532 solution (pH 9.0). Contamination of rainbow trout milt with urine led to dose-dependent decrease in seminal plasma osmolality, from 301 3 to 203 2 mOsm/kg, for 0 and 30% contamination, respectively. Contamination with urine signicantly reduced the ability of rainbow trout spermatozoa to be activated at pH 6.5 after 2 and 4 h of incubation. On the other hand, no changes of motility in pH 9.0 saline were observed at the same time. Our results suggest, that the ability of spermatozoa to activate motility at pH 6.5 can reect sub-lethal changes of rainbow trout spermatozoa after urine contamination. 2011 Elsevier B.V. All rights reserved.

Article history: Received 15 June 2011 Received in revised form 5 December 2011 Accepted 13 December 2011 Available online 29 December 2011 Keywords: Rainbow trout Sperm Motility Urine Osmolality

1. Introduction Contamination of milt with urine is of major importance regarding the sperm quality of freshwater sh. 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 sh 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-specic 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
Corresponding author. Fax: + 48 89 535 7421. E-mail address: a.ciereszko@pan.olsztyn.pl (A. Ciereszko). 0044-8486/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.12.023

suggestion. We collected urine and milt separately using a catheter and then experimentally introduced urine to milt at 1030%. 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. 2. Materials and methods 2.1. 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. 2.2. Effects of urine contamination on sperm motility Semen samples from each individual were mixed with 10, 20 and 30% of urine. The nal volume of suspensions was 200 l. Samples were incubated at 4 C. At 0, 2 and 4 h of incubation samples of

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Motile sperm (%)

sperm suspensions (1 l) were withdrawn and sperm motility at pH 6.5 was measured. After 4 h samples were centrifuged for osmolality measurement. 2.3. Analytical methods All measurements were made in duplicate for each n. Motility data are therefore the mean from two measurements. Sperm motility at pH 6.5 and pH 9.0 for each sperm sample were examined with Computer Assisted Sperm Analysis (CASA) using the Hobson Sperm Cell Tracker (Hobson Vision Ltd, Baslow, UK) as described by Dietrich et al. (2005). Sperm suspensions were activated at a dilution ratio of 1:250 with either MES-NaOH pH 6.5 (2-(N-morpholino) ethanesulfonic acid; Sigma, 30 mOsm/kg) or solution D532 (1 mM CaCl2, 20 mM Tris, 30 mM glycine, 125 mM NaCl, pH 9.0, 271 mOsm/kg; Billard, 1992) supplemented with 0.2% bovine serum albumin. The following CASA parameters were registered: the percentage of all motile sperm and curvilinear velocity (VCL). The percentage of motile sperm in Hobson Sperm Tracker is dened by the number of motile sperm within the analysis eld, divided by the sum of the motile plus immotile sperm within the analysis eld (Kime et al., 2001). Video recordings (two copies per sample) were made using a microscope with a 10 negative phase objective and a Sony CCD black and white camera (SPT-M108CE). The control samples were semen from each individual without urine. Sperm samples were centrifuged twice (supernatant from rst centrifugation was centrifuged again to ensure that no sperm cells are present in seminal plasma) at 8000 g to obtain seminal plasma and urine was centrifuged once at 8000 g. Osmolality was measured using a Vapor Pressure Osmometer 5520 (WESCOR, Logan, USA). Sperm concentration was measured using computer-aided uorescent microscopy (Nynca and Ciereszko, 2009). 2.4. Statistical analyses Data are presented as means SD. All analyses were performed at a signicance level of 0.05. For statistical procedures data percentages were transformed by arc-sin square root transformation. Data were subjected to one- way and two-way repeated measurements ANOVA followed by LSD Fisher's test for post hoc comparisons of means. Statistica (Version 9.0, 2007, StatSoft, Inc., Tulsa, OK, USA) and GraphPad Prism software (GraphPad Software Inc, San Diego, CA, USA) were used for statistical calculations. 3. Results 3.1. 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 10 9 spermatozoa x ml 1 and osmolality of pooled urine

100
0

80 60 40 20 0 0 2 Time of exposure (h) 4

10 20 30

Fig. 2. The effect of time of exposure and urine contamination on rainbow trout sperm motility rate which can be activated in D532 at pH 9.0 (n = 5).

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 between seminal plasma osmolality and the percentage of urine contamination was calculated as y = 3.20x + 93.21, r 2 = 0.99, p b 0.01. 3.2. Effect of urine concentrations and time of exposure on the sperm motility rate activated at pH 6.5 At time 0, the addition of urine doses to semen did not affect the motility rate of sperm activated at pH 6.5 with the exception of sperm samples containing 10% urine (Fig. 1). However, after 2 h of storage, a signicant dose-dependent decline in the values of the percentage of sperm motility was observed for all semen samples with the additions of urine. Exposure of milt to 10, 20 and 30% urine contamination resulted in 44, 48 and 68% decrease in percentage of motile sperm, respectively. After 4 h of storage no further major changes in percentage of sperm motility were observed. Curvilinear velocity was similar for all variants (range 68.8 m/s 97.7 m/s) with the exception of 4 h incubation and 30% of urine contamination, where lower (P b 0.05) values were recorded (68.8 9.7 m/s compared to 87.5 8.8 m/s after 4 h incubation of samples without the addition of urine). 3.3. Effect of urine concentrations and time of exposure on the sperm motility rate activated at pH 9.0 No differences at any time point were noticed between the percentage of sperm motility rate activated at pH 9.0 of control samples and samples with all tested concentrations of urine (Fig. 2). 4. Discussion In this study, we have demonstrated that experimental contamination of rainbow trout milt with urine can lead to signicant dosedependent decrease in seminal plasma osmolality. Such contamination signicantly 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 low values of osmolality were reported. For example, Munkittrick and Moccia (1987) recorded average values as low as 169, 131, and 116 mOsm/ kg for milt samples collected in February, March, and April, respectively. Similar low values were also recorded by other authors

80

ax ab x ab x a x by ax by bc z cy cy

Motile sperm (%)

60

bx
40

0% 10% 20% 30%

by

20

0 0 2 4

Time of exposure (h)

Fig. 1. The effect of time of exposure and urine contamination on rainbow trout sperm motility rate which can be activated at pH 6.5 (n=5). Values with different superscripts are signicantly different (Pb 0.05) among urine concentrations (a, b, c) or exposure time (x, y, z).

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(Ciereszko et al., 1996). Lahnsteiner et al. (1998) recorded 290.9 77.6 mOsm/kg for seminal plasma obtained from rainbow trout semen collected with catheter. Aas et al. (1991) recorded of a range from 117 to 320 mOsm/kg for Atlantic salmon seminal plasma osmolality. The most obvious explanation for a decreased osmolality of semen is urine contamination introduced during stripping, since the osmolality of urine itself is very low. This suggestion is indirectly supported by observations of an increased osmolality of semen in relation to the sequential collection of milt reported by Dietrich et al., 2005 (urine is usually released rst during stripping) and high values of osmolality of semen samples collected with a catheter (Glogowski et al., 2000). Surprisingly, to the best of our knowledge osmolality of seminal plasma in response, to experimental milt contamination of rainbow trout semen with urine has never been studied. Our study provided for rst time data, demonstrating to what extent seminal plasma osmolality would be decreased after urine contamination. It is clear from our data, that in practical conditions of rainbow trout semen collection by stripping, contamination of semen with urine is quite high, and osmolality of about 200 mOsm/kg reects 30% contamination, so values around 140 mOsm/kg would indicate that urine comprises as much as half of the semen volume. These values can be slightly different due to the variability in urine composition. Osmolality of 300 mOsm/kg should be recognized as typical for uncontaminated semen. Although detrimental effects of urine on sperm activation at pH 6.5 seem to be mainly related to a decrease in semen osmolality, it should also be emphasized that other factors can also add to such effects. Urine consists of different inorganic and organic compounds and has a specic pH (within a range 7.47.8, Bucking et al., 2010). Among inorganic substances, potassium, sodium, calcium and magnesium are present in urine of rainbow trout (Chowdhury and Wood, 2007), as well as in urine of other sh species (Linhart et al., 2003). All these ions and pH can modulate sperm motility (Alavi and Cosson, 2005, 2006). Among organic components of rainbow trout semen ammonia is present in urine of rainbow trout (Chowdhury and Wood, 2007). For mammals it has been demonstrated that the increased levels of ammonia lower the pH, leading to loss of sperm motility (Kim and Kim, 1998; Tareq et al., 2008). Further studies are necessary to evaluate if above urine parameters alone, or through synergistic action, can participate in the mechanism of negative action of urine on rainbow trout spermatozoa. In our study, contamination with urine did not affect the percentage of sperm motility measured at the optimal conditions recommended for fertilization in rainbow trout (Billard, 1992). These results do agree with existing knowledge indicating that the milt of rainbow trout collected by stripping (thus most likely contaminated with urine) can be stored for several days or a few weeks (Stoss, 1983), although high individual variability in the sperm quality is observed (Nynca et al., unpublished information). It can be therefore suggested, that spermatozoa of rainbow trout can somehow compensate for the detrimental effects caused by the decline in osmolality caused by urine contamination. It is possible, that this compensation can be a species-specic feature of rainbow trout; the detrimental effects of urine seem to be more pronounced for other species such as tench (Linhart et al., 2003), carp (Poupard et al., 1998) and turbot (Dreanno et al., 1998). It has to be emphasized that a decrease of the osmotic concentration of the activation medium is typically necessary for motility initiation in freshwater sh. Further studies are necessary to identify the mechanism responsible for the maintenance of rainbow trout spermatozoa's ability to be activated at pH 9.0 after urine contamination. Our results clearly demonstrate that the exposure of rainbow trout milt to urine contamination impaired the ability of the spermatozoa to be activated at pH 6.5. A decrease of the ability of sperm to be activated at pH 6.5 after contamination with urine provided direct evidence to support our hypothesis, that previously described variability

of rainbow sperm motility at pH 6.5 is related to urine contamination (Ciereszko et al., 2010). Since the ability for the activation of sperm motility at pH 6.5 seems to be a feature of uncontaminated semen it can be suggested that a loss of this ability in the contaminated milt samples reects sub-lethal damage to membranes and/or motility apparatus. The nature of such damage is unknown at present. It remains to be established, if ability of sperm to be activated at pH 6.5 can reect sperm quality, for example regarding tness of the semen to withstand a serious challenge, such as short-term storage and cryopreservation. This suggestion is supported by the results of Malejac et al. (1990) and Lahnsteiner et al. (1996) who demonstrated that rainbow trout semen tness with regard to cryopreservation is related to semen osmolality. Recently we obtained evidence that sperm motility rates of short-term (8 days) stored rainbow trout semen are higher for samples characterized by ability to be activated at pH 6.5 (Nynca et al., 2011). This suggests that the testing of rainbow trout spermatozoa activation at pH 6.5 can be used for evaluation of rainbow trout sperm quality and to predict semen usefulness for short-term storage. The mechanism responsible for a reduction in sperm motility at pH 6.5 in relation to urine contamination is unknown at present. Woolsey et al. (2006) have demonstrated that the percentage of sperm motility is related to the pH of pre-incubation mixtures. In their experiment, sperm suspensions incubated for 2 h at pH 7.1, contrary to those incubated at pH 8.5, lost the ability of motility activation at pH 6.5. Ingermann et al. (2008) suggest that motilityassociated processes are located at the external surface of the sperm. It remains to be established, if urine effects on sperm motility described in this study are related to modications of sperm suspension pH and/or changes in the surface of the sperm. In conclusion, our results clearly demonstrate that exposure of rainbow trout milt to urine contamination impaired ability of the spermatozoa to be activated at pH 6.5. It is possible, that this ability can reect sub-lethal damage of rainbow trout spermatozoa after urine contamination, and can be potentially used as a sperm quality parameter. Further studies are necessary to test if such a parameter can be useful for the selection of good-quality semen samples for short-term and long-term storage. Acknowledgements This study was supported by funds appropriated to Institute of Animal Reproduction and Food Research. References
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