EVALUATION OF CASA SYSTEM FOR PREDICTING THE MOTILITY OF ZEBRA MUSSEL (DREISSENA POLYMORPHA PALLAS) SPERMATOZOA

Joanna Białkowska, Grzegorz J. Dietrich, Wiesław Demianowicz, Jan Glogowski

ABSTRACT

A computer – aided semen analyzer (CASA) was used for monitoring some motility parameters of zebra mussel sperm after spawning for up to 1,5h at 22°C and after 24 hours of storage at 8°C. The mean percentage of motility (MOT) for sperm shortly after spawning was 70ą2.0 %, VSL = 31.8ą3.0 µm s^-1, and VCL= 110.2ą6.4 µm s^-1. The motility expressed as LIN was 27.7ą2.0 %, STR=44.5ą4.8 %, ALH=8.7ą2.0 µm, BCF=6.8ą2.0 Hz. The data demonstrate a consistent decrease in VSL (12.8ą 4.0), VCL (57.4ą 5.7), LIN (15.8ą 3.0) and ALH (1.1ą 0.23) values after 24 hours. No significant differences were found for % of MOT, BCF and STR parameters values during the time of experiment. We found CASA analyzer as a proper tool helpful to discerning the quality of zebra mussel semen samples.

Key words: Dreissena polymorpha, sperm motility, CASA..

INTRODUCTION

The zebra mussel, Dreissena polymorpha, is a freshwater, highly fecundative bivalve, accidentally introduced into inland waters of North America from Europe in the mid-1980s [6]. This biofouling pest species has a highly active filtering ability and forms large aggregation on hard substrates with high population densities, which causes important changes in ecology and water clarity. However, the biggest phenomenon is its external fertilization and long duration of sperm motility. As an approach to understanding better the mechanisms involved in its prolific reproduction and gamete behavior, motility and some other motility parameters were determined using the CASA system. Generally there are some papers that deal with North American and European zebra mussel sperm motility parameters [4, 11, 12] but none on detailed description of European D. polymorpha sperm kinematics using CASA.

The computerized analysis system can quantify a range of parameters of sperm motility. This provides a rapid and objective method for assessing the motility of many species, especially mammalian and fish sperm. Zebra mussel sperm is ideal to detect its character of motility as it lasts almost three hours at room temperature and 24 hours at 4°C [4, 18]. Unlike most fish species which can be motile only a few minutes or even seconds [3], zebra mussel sperm is exceptional and ideal to work with. Adaptation of the CASA system to describing the quality of zebra mussel sperm can prove that precise analysis of sperm kinematics can be used to analyze other aspects of mussel sperm motility/quality in future toxicological, fertility tests and other experiments.

Objective of the present study: to determine the basic motility parameters of zebra mussel sperm shortly after spawning and during storage.

MATERIAL AND METHOD

Obtaining zebra mussel sperm:

Animals were collected from Lake Kortowo (Poland) and kept in an aquarium with tap water non-stop arreated (12 - 16°C). Sperm were obtained through the external application of 0.5 mM serotonine in fresh water for 15 minutes (FW, pH=7.6) [18] as described by Ram et al. [16].

The number of sperm per milliliter from four specimens were determined using a hemocytometer [1]. Motility analysis for sperm samples from the same males was performed using the Hobson Tracking System (Hobson Vision Ltd.) with negative phase contrast microscopy, and a Sony CCD black and white video camera (SPT-M108CE), which automatically corrects changes in light intensity.

Program settings for the image analysis at x 10 objective magnification were: search radius=9.69 mm; predict = off; video = pal; aspect = 1.49; refresh time = 15 s; threshold +16/-100; filter weightings 1=2, 2=2; 3=2; 4=2, image capture rate = 50 Hz.

The analysis was performed immediately after obtaining sperm. To prepare sperm for visualization and counting ICN Multitest slides (12-cell) and cover slips (thickness No 1) were used.

Hobson Tracker simultaneously assessed fifteen parameters [8], but for brevity only seven representative: VSL - straight line velocity (µm s^-1), VCL – curvilinear velocity (µm s^-1) ALH - amplitude of lateral head displacement of the sperm head (µm), BCF – beat cross frequency (Hz), LIN – linearity (%), STR – straightness (%) and MOT - % of motile sperm were analysed.

Preparation for analysis

1µl of diluted fresh / stored sperm was pipetted onto multitest slide and gently covered with cover slip. About 30-50 spermatozoa on the screen were observed during the first 30 seconds. The motility of semen from four specimens expressed as MOT, VSL, VCL; BCF, ALH, LIN and STR have been assessed at 0; 0.5 and 1.5 hours from obtaining (22°C) and after storage for 24 hours at 8°C.

Statistical analysis

Motion characteristics of D. polymorpha spermatozoa from multiplied samples were recorded as means ą SEM. Differences among the means compared between all groups (0-24h) were analyzed using a one-way ANOVA and Tukey post-hoc test. All analyses were performed at significance levels of 0.05 using GRAPHPAD PRISM v. 2.0.

RESULTS

Motility study

D. polymorpha spermatozoa remained active for almost 1.5 hour in room temperature and almost 24 hours stored at 8°C. The mean % of motility for sperm shortly after spawning was 70 ą1.93% (Fig. 1A) and did not change significantly during 24 hours (p£0.05).

After 24 hours, sperm exhibited 46.5 ą 4.3% of motility. The initial value of VCL for D. polymorpha sperm exhibited 110.2 ą 6.4µm s^-1(Fig.1B), VSL was 31.8ą3.0µm s^-1 (Fig.1C) at the beginning of the experiment. Changes in VCL and VSL had generally a similar trend which appeared by decreasing values after 24 hours of the experiment (p£0.001 for VCL and p£0.05 for VSL) (Fig. 2A, 2B). The same alternations were noted for ALH, LIN where significant differences were seen after 24 hours of experiment (p£0.05) (Fig. 2C, 2D).

Motility expressed as BCF (Fig.1F) and STR (Fig.1G) did not change during the time of the experiment. There was a positive correlation for values of parameters measured after 24 hours of sperm storage at 8°C between VCL and BCF (r²=0.9, p£0.05) and VSL (r²=0.99, p£0.01). The MOT correlated with STR (24h, r²=0.97, p£0.05).

DISCUSSION

This paper provides a more detailed description of the characteristics of Dreissena polymorpha sperm motions. This is some of the first data describing European D. polymorpha sperm kinetic using computerized analysis - CASA. In 1995 Mojares et al. [12] described the motility of North American zebra mussel spermatozoa using CASA comparing the motility of sperm stripped from gonads and after spawning. The motion alternations of D. polymorpha sperm as a response to attractants have already been analyzed [11]. Ciereszko et al. [4] also characterized the duration of movement, effects of cations and pH on sperm motility of European mussels by expressing results as a percentage of motile cells by subjective microscopic examination.

It is very difficult and almost impossible to assess kinematics characteristics of sperm by subjective microscopic examination. CASA is an ideal instrument to detect all these parameters and describe the character of sperm motility. Centola [2] demonstrated that CASA gives much more detailed results that are less charged by errors then the manual microscopic observation. Describing such parameters as curvilinear /straight-line velocity or others may predict some distinct changes in motility character whereas percentage of sperm motility (MOT) assessed by CASA or microscopic observations can remain unchanged. Sperm motility is an essential parameter in the examination of sperm quality and in the establishment of a correlation between sperm quality and fertility [23].

Our data indicate lower values of D. polymorpha sperm motility expressed as MOT- 70% (Fig.1A), VCL-110µm s^-1 (Fig.1B), VSL-31µm s^-1 (Fig.1C), than those reported by Mojares et al [12]. D. polymorpha sperm analyzed by these authors were > 90% motile, and exhibited VCL and VSL exceeded 150µm s^-1 and 60µm s^-1, respectively, whereas sperm stripped from ripe gonads were less motile (30 %) and had slower VCL (58µm s^-1).

Miller et al. [11] characterized zebra mussel sperm movements during attractant application (extracts from oocytes) and before or after injection near a chemo-attractant source. He also found that sperm had a high level of motility > 90% and exhibited linear velocities of approximately 200µm s^-1.

As said Mojares et al. [12] in their investigations MOT and VCL of zebra mussel spermatozoa is similar to cat (79% and 178µm s^-1) and dog (93%, 159µm s^-1) [7, 19]. Human and rat sperm exhibited a lower kinematic value: human sperm - 38% of 52 µm s^-1, rat sperm 58% of 148 µm s^-1 [5, 20].

Kinematic analysis of our data revealed that sperm exhibited lower motion characteristics (velocities, LIN and ALH) after 24 hours of storage at 8°C, whereas % of motility, BCF, STR did not change during time of experiment. The 24 hours storage at 8°C motility of D. polymorpha spermatozoa does not decrease significantly in our data whereas sperm stored for one day at room temperature caused a total decline in % of motility.

Ciereszko et al. [4] have not found significant differences in percent of motility or duration between European and North American zebra mussel spermatozoa populations after 3h storage at room temperature. A decrease in sperm motility of North American mussels was observed after 24 hour storage at room temperature (down to zero percent), whereas on ice both European and American populations had almost 60% and 70%, respectively. Ravinder et al. [17] reported that the storage of carp semen at room temperature (22°C) or in the presence of KCl (22°C ) with the initial motility ranged from 40-90% led to a steady, sharp decline in % of motile sperm. In marine teleosts, VSL decreased progressively from 160 - 20µm s^-1 with time following activation [15, 22]. In King’s et al. [10] experiments, there was a significant positive correlation between VSL, LIN, BCF and sperm motility.

In our preliminary experiment there was a significant positive correlation between BCF, VSL and VCL after 24 h. The MOT correlated only with STR (24h), but a study on some toxicological and especially fertility tests may give some different responses in future experiments.

Zebra mussel sperm is approximately 4 µm long, measuring from the top of the conical acrosome to mitochondria [12, 24] with the midpiece consisting of 4-5 spherical mitochondria producing the energy required for sperm movement and 50 µm long flagellum that propagates the propulsive waves initiated at the midpiece. If in the initiation phase the flagellar waves are near the head and small in amplitude, the sperm head will have a small lateral movement and a more pronounced forward movement [14]. Zebra mussel sperm had its amplitude lateral head displacement – 8.7µm (Fig.1D), beat cross frequency – 6.8 Hz (Fig. 1F) and motility - 70% (Fig.1A). There was no significant correlation between these parameters and no changes for BCF and MOT during the time of experiment as was mentioned.

Toth et al. [21] reported that changes in sperm motion endpoints can reflect sperm vigor and swimming patterns and may be an indication of changes in the sperm that are metabolic and structural.

CASA provides a rapid quantitative assessment of the quality of fish sperm and may predict fertilization success [9]. Moore and Akhondi [13] worked with rat sperm have shown that the fertilizing capacity of sperm correlates with the decline in straight-line velocity, but it is still not clear how some spermatozoa motility parameters may relate to a decrease or increase in fertility. It is conceivable that a reduction in sperm motility will lead to a subsequent decrease in fertilization rate.

In our preliminary experiments there was a significant decrease in the fertility ratio when the % of motility decreased (unpublished data). There could be other constant and sensitive parameters to predict fertility, and CASA can be useful. Further investigation could prove that CASA and its ability to evaluate motility parameters as VSL or others may be related and predict fertilization rates.

The CASA system can be adopted to detect the influence of such factors such as water pH, temperature, motility inhibitors and toxicity test to assess the potential hazards of environmental pollutants on zebra mussel sperm survival and its reproduction. Kinematics analysis of sperm motility may also help to determine whether mussels can reproduce in brackish water estuaries and provide valuable insights into controlling Dreissena polymorpha encroachment [12].

The different CASA instruments have demonstrated high levels of precision and reliability using different sperm classification methodology. Their availability provides a great tool to objectively compare sperm motility and morphology and to improve our knowledge and ability to manipulate spermatozoa [23]. CASA system can serve as an objective, quick and quantitative assessment of the effects of time on sperm motility of Dreissena polymorpha. Further experiments are needed to compare motility parameters and fertility rate because the relation between alternations in sperm trajectory and other changes of motility are still not understood clearly and further investigations should be done.

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Grzegorz J. Dietrich
Semen Biology Group
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences
Tuwima 10, 10-747 Olsztyn 5, P.O. Box.55, Poland
dietrich@pan.olszyn.pl

Wiesław Demianowicz, Jan Glogowski
Molecular Andrology Group
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences
Tuwima 10, 10-747 Olsztyn 5, P.O. Box.55, Poland

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