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Male Reproductive Tract |
Laboratorio di Fisiologia Generale e Comparata Dipartimento di Scienze e Tecnologie Biologiche e Ambientali,2 Università di Lecce, 73100 Lecce, Italy
Marine Aquaculture and Fisheries Research Centre,3 Frigole-Lecce, 73100 Lecce, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-irradiated [3], or cryopreserved [4, 5] spermatozoa were not fertile. The value of motility for predicting fertility is also questionable because of the subjectivity of the technician performing the analysis and the short duration of motility following activation. Fertilizing capacity is the most conclusive test of sperm quality. However, the use of this marker of sperm quality is laborious and requires the availability of eggs [6]. Until now only a few researchers have investigated biomarkers for semen fertilization capacity. In fresh semen of the Atlantic salmon (Salmo salar), sperm density and sodium, potassium, glucose, and sodium to potassium ratios were positively correlated with fertilization rate [7, 8]. In rainbow trout (Oncorhynchus mykiss), sperm density and aspartate aminotransferase activity were correlated with fertilization rate [9]. The postthaw fertilization rate of rainbow trout semen was correlated with pH, triglycerides, ß-D-glucuronidase activity, lactate dehydrogenase activity, and spermatozoan acid phosphatase and adenylate kinase activity [10]. The fertilization rate of rainbow trout semen has been described by three multiple regression models [11]. The first included parameters of seminal plasma (pH, ß-D-glucuronidase activity, and total lipids), the second included sperm metabolism parameters (malate dehydrogenase activity, respiration activity, aspartate aminotransferase activity, and total lipids), and the third included sperm motility parameters (motility rate and total swimming velocity). However, generalizations cannot be made from these data because the studies concern different species. The aim of this study was to investigate the possible relationship between sea bass semen parameters and fertilization rate to identify simple and cost-effective tests that would replace conventional motility and fertility evaluation assays, using both fresh and frozen-thawed sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The D-Trp6 LHRHa (D-Trp6-Pro9-NEt-LHRH analogue) was purchased from Sigma Chemicals (St. Louis, MO).
Gamete Collection
The study was carried out on sexually mature broodstock of reared sea bass males (3 yr old, length 25–30 cm, weight 200–250 g). The experiments were carried out during the reproductive period, which in Lecce, Italy runs from 7 January to 27 February. The broodstock was kept in an indoor tank at a density of 0.6 kg/m3. The broodstock tank was provided with seawater constantly at a rate of 1 L/sec, and compressed air was provided through airstones. Sea bass were fed daily ad libitum with pellets, and fresh trash fish were provided once weekly. The water temperature ranged between 13.5 and 15.5°C, and the salinity was 34.2%. When handled, fish were anesthetized using 0.1 ml/L 2-phenoxy ethanol. For each experiment, semen was collected by applying gentle pressure to the testes and sperm ducts to obtain a drop of milt on the previously cleaned gonophore area. Urine and semen that may have been contaminated with urine (checked by color changes and viscosity variations) were discarded. Sperm was collected in 2-ml syringes and stored at 4°C on ice for 
15 min. Sperm motility was evaluated immediately after semen collection. Each sample was divided in two aliquots. One aliquot was diluted at a ratio of 1:3 in nonactivating medium (3.5 mg/ml NaCl, 0.11 KCl, 1.23 mg/ml MgCl2, 0.39 mg/ml CaCl2, 1.68 mg/ml NaHCO3, 0.08 mg/ml glucose, 10 mg/ml BSA, pH 7.7) and used for the analysis. The other aliquot was used for cryopreservation. For egg collection, females were isolated at 13°C in individual tanks (1 m3). Eggs were collected by stripping 72 h after a single injection of 10 µg/kg of D-Trp6 LHRHa. The quality of the ova was estimated from their morphological features observed under a dissecting microscope [12].
The animal studies were approved by the Animal Care and Treatment Committee of the Università di Lecce.
Cryopreservation of Semen
The freezing protocol developed by Fauvel et al. [2] was applied for semen cryopreservation. Semen samples were diluted in Mounib medium (10.01 mg/ml KHCO3, 1.99 mg/ml reduced gluthatione, 42.78 mg/ml sucrose, 10 mg/ml BSA, 10% dimethyl sulfoxide, pH 7.8, osmolality 310) at a ratio of 1:3 (semen:Mounib medium) and placed into 0.25-ml straws. Straws were placed horizontally on a tray 6.5 cm above the surface of the liquid nitrogen. After a freezing period of 15 min, straws were plunged into the liquid nitrogen for storage. For thawing, straws were immersed for 5 sec in a warm water bath (35°C).
Fertilization Assays
The fertilizing ability of fresh and frozen-thawed semen was evaluated using the following experimental protocol. Fresh sperm were prepared by direct dilution to 1:10 (v/v) in nonactivating medium. For cryopreservation, sperm were diluted 1:3 (v/v) in Mounib medium before freezing and 1:3.3 (v/v) in nonactivating medium at thawing (resulting dilution of thawed sperm was similar to that of fresh sperm). Triplicate 5-ml batches of ova were inseminated with 150 µl of diluted sperm. Frozen sperm samples were thawed and diluted individually just before insemination to avoid any possible decrease of fertility due to the postthaw delay. Fertilization was triggered by the addition of 2.5 ml seawater (38 g/L, 13°C). Sperm was allowed to fertilize eggs for 3 min. Inseminated eggs were then transferred into vials containing 100 ml of seawater where early development took place. After 3 h, the vial contents were poured onto a net, and both floating and sinking eggs were placed onto a counting plate. The resulting fertilization rate was assessed under a dissecting microscope based on examination of 200 randomly chosen eggs. Eggs were assumed to be fertilized when they exhibited at least the four-cell stage, which occurred 3 h after fertilization at 13°C.
Eosin Test
The eosin-Y staining test (0.5% w/v) was performed by mixing 10 µl of semen with 10 µl of the stain on a microscope slide covered with a 22- x 22-mm coverslip. A total of 100 spermatozoa were then counted within 2 min after the addition of the stain. The results were expressed as the percentage of unstained (live) sperm.
Preparation of Seminal Plasma and Spermatozoa
The protocol of Lahnsteiner et al. [10] was adapted to sea bass milt and used to separate the seminal plasma from the spermatozoa. Each semen sample was centrifuged at 800 x g for 10 min at 4°C. The supernatant (seminal fluid) was centrifuged again (800 x g for 10 min at 4°C) to avoid possible contamination by spermatozoa. The pellet containing the spermatozoa was resuspended in the nonactivating medium for sea bass sperm at a ratio of 1:5, centrifuged (800 x g for 10 min at 4°C), and resuspended in the nonactivating medium. This sperm preparation was divided into two subsamples; one was used to determine enzymatic activities and the other was used to measure metabolite content.
For the preparation of crude enzyme extracts, the spermatozoa were washed twice in the nonactivating medium and diluted with Tris buffer (100 mM, pH 7.5) containing 2 mM EDTA and 0.1% Triton X-100 at a ratio of 1:4, mixed vigorously, and plunged into liquid nitrogen. Samples were thawed at room temperature, further diluted to a ratio of 1:2 with the same buffer containing 2 mM EDTA but no Triton X-100, and centrifuged for 15 min at 1000 x g. The supernatants were used for enzymatic analysis.
Metabolites were extracted with cold (4°C) 3 M perchloric acid, which was added to the sperm suspensions at a ratio of 1:4. After 15 min, the extract was centrifuged (15 min at 1000 x g), the pellet was discarded, and the supernatant was neutralized with 2 M KOH and used for the analysis.
Determination of pH, Osmolality, and Ion Concentration
The pH of the seminal plasma was determined with a semimicro electrode (Roebling, Cambridge, U.K.) using an external reference electrode (3.0 M KCl), the osmolality was measured using an automatic osmometer (5520 VAPRO, Delcon, Milano, Italy), and the sodium and potassium concentrations were measured using a digital pH/ion meter (Metrohm, Herisau, Switzerland) with ion-selective electrodes.
Determination of Metabolite Concentrations
Triglyceride concentrations were determined by using the enzymatic method described by Bucolo and David [13], ATP concentration was assayed uisng the enzymatic method described by Adams [14], and protein concentrations were determined using the Lowry procedure [15].
Determination of Enzyme Activities
Enzymatic activities were measured using the methods described by Bergmeyer [16] that were adapted to sea bass semen for aspects of incubation time, pH, substrate concentration, and optimal reaction temperature. For aspartate aminotransferase, fixed incubation time was 1.30 h, pH was 7.5, substrate concentrations were 165 mM DL-aspartate and 1.5 mM 2- oxoglutarate, and optimal reaction temperature was 20°C. For malate dehydrogenase, pH was 7.4; substrate concentrations were 40 mM DL-aspartate, 1 mM 2-oxoglutarate, and 0.2 mM NADH; and temperature was 20°C. For isocitrate dehydrogenase, fixed incubation time was 5 min, pH was 7.8, substrate concentrations were 0.007 mM LD-isocitric acid and coenzyme 0.5 µM NADP, and temperature was 20°C.
Respiration Measurements
For oxygen measurements, an oxygraph (Oxygraph Measurement System, Hansatech Instruments Ltd, Nortfolk, U.K.) supplied with a platinum cathode, a silver anode, and a high-sensitivity 12-mm Teflon membrane was used. The bath stirrer unit was maintained at a constant temperature (21°C) with a thermoregulated controlled circulating system, and stirring was constant at 800 rpm in all experiments.
The reaction vessel was closed with an exactly fitted plunger containing the electrode. The bottom of the plunger had an escape slot for removal of air bubbles and for injection of additives. The electrode system was calibrated with oxygen-free buffer (by addition of sodium ditioniote) and with air-saturated buffer. All solutions were equilibrated to 21°C. The experiments to measure respiration rate in motile semen followed the following design. Seawater (0.8 ml) was placed in the investigation chamber, and 0.2 ml semen (fresh sperm diluted 1:3 in nonactivating medium, frozen-thawed sperm diluted 1:3 in Mounib medium) was added. The reaction vessel was immediately closed with the plunger containing the electrode, the magnetic stirrer was switched on, and air bubbles were removed through the slot. The whole operation took about 20 sec, and another 10 sec elapsed until the response of the electrode was linear. Therefore, all measurements of respiration for motile spermatozoa started 30 sec after initiation of motility. Measurements were made for 5 min. During this period, the decrease in oxygen concentration was linear in all samples. To determine whether the oxygen consumption could be ascribed to mitochondrial activity rather than to no specific oxygen binding, sperm respiration rate was also measured in the presence of 5 mM KCN (concentration in assay), which inhibits cytochrome c respiration. KCN was added in seawater before sperm was injected into the respiration chamber.
Statistical Analysis
The values of sperm metabolites were referred to the protein concentration of the sperm fraction, and enzymatic activities extracted from spermatozoa were referred to the protein concentration of the crude enzyme extract. The metabolites and enzymes of the seminal plasma were referred to a defined volume of seminal plasma.
The means of continuous variables such as fertilization rate and metabolite and enzyme values in fresh sperm were compared with those measured in frozen-thawed sperm using a two-tailed Z-test. Differences were considered significant at P = 0.01.
The parameters of sperm metabolism and seminal plasma were tested by evaluating correlations with the fertilization rate using simple regression analysis with SYSTAT 5.0 for Macintosh or using square relationship analysis with ANA-DAT 1.0 for Windows 98.
To perform the multiple regression analysis, the following assumptions were tested. Independence of independent variables was tested with the bivariate correlation of Pearson coefficients, normal distribution of variables was tested with the Shapiro-Wilks test, and homoscedasticity was tested with the Barlett test.
The multiple regression analysis was carried out using StatGraphics Plus 4, and the significance levels are listed with the results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sea bass sperm metabolism was characterized by measuring the activity of key enzymes and metabolite concentrations in some important metabolic pathways (Table 1). In the seminal plasma, the concentration of inorganic and organic compounds and the activity of a lytic enzyme (ß- D-glucuronidase) were measured. The values of chemical and biochemical parameters reported are the means of determinations carried out during two different reproductive seasons (2002 and 2003). We pooled these data to get a more accurate evaluation of the parameters.
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The effect of cryopreservation on sperm metabolism was determined by measuring some of the parameters both before and after cryopreservation. Most of the measured parameters were not affected by the freezing-thawing procedure (Table 1). In addition, eosin uptake results were similar in fresh and frozen-thawed sperm (75%–80%). Only malate dehydrogenase activity and intracellular ATP concentration were significantly higher in the cryopreserved samples. However, the pH of seminal plasma was significantly lower after freezing.
Relationship of Sperm and Seminal Plasma Parameters and Fertilization Rate in Fresh Semen Samples
Parameters of sperm metabolism and seminal plasma were tested by evaluating correlations with the fertilization rate using simple regression analysis and square relationship analysis. To perform these analyses, data obtained during the 2003 reproductive season were used. The mean values of these parameters were not significantly different from those reported in Table 1.
Among the sperm metabolites and enzymes, only ATP concentration and aspartate aminotransferase activity showed significant linear correlations (P < 0.0001) with fertilization rate (Fig. 1). The simple regression functions describing these relationships are shown in Figure 1. In addition the calculation of the partial correlation coefficient revealed that ATP and aspartate aminotransferase were not correlated (Pr = –0.323). Malate dehydrogenase activity and sperm triglyceride concentration had a quadratic relation with fertilization rate: R2 = 0.31, P < 0.001 for malate dehydrogenase; R2 = 0.28, P < 0.01 for triglyceride concentration (Fig. 2).
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Among seminal plasma chemical and biochemical parameters investigated, only ß-D-glucuronidase activity and potassium concentration had a significant linear relation (P < 0.01) with fertilization rate (Fig. 3). The simple regression functions are shown in Figure 3. potassium concentration and ß-D-glucuronidase activity were not correlated (Pr = 0.241).
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Sperm and seminal plasma parameters that had a linear correlation with the fertilization rate were used in multiple regression models to predict the fertilization ability of semen samples. The first model includes the sperm ATP concentration (x1) and aspartate aminotransferase activity (x2): y = (14.1 ± 5.8)x1 – (27.4 ± 12.6)x2 + (61.4 ± 12.5) (R2 = 0.68, P < 0.001, F = 8.77). The second model includes a seminal plasma ß-D-glucuronidase activity (x3) and potassium concentration (x4): y = –(32.0 ± 12.1)x3 – (0.6 ± 0.2)x4 + (99.2 ± 6.5) (R2 = 0.66, P < 0.001, F = 15.68). The third model includes ATP (x1), aspartate aminotransferase (x2), and ß-D-glucuronidase (x3): y = (10.8 ± 5.4)x1 – (40.9 ± 10.6)x2 – (27.9 ± 10.3)x3 + (75.11 ± 11.2) (R2 = 0.61, P < 0.001, F = 10.08), but this model does not include potassium becuase a linear relationship exists between intracellular ATP and seminal plasma potassium concentration (Pr = –0.973). Results indicate that semen fertilization rate could be best predicted by the first multiple regression model, which includes the sperm parameters.
Relationship of Sperm and Seminal Plasma Parameters and Fertilization Rate in Cryopreserved Semen Samples
Because sperm ATP concentration and seminal plasma ß-D-glucuronidase activity among the tested parameters produced the highest correlation coefficients, we also investigated their relationship with fertilization rate in frozen- thawed samples. Results shown in Figure 4 reveal that these parameters have a linear relationship with fertilization rate after the freezing-thawing procedure. The simple regression functions also are shown in Figure 4. The functions were similar in fresh and cryopreserved samples.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Until now, the effect of cryopreservation on sea bass sperm has been evaluated based on motility and fertilization rate [2] and recently on DNA integrity [23]. Previous studies carried out on semen of freshwater species [6, 11, 24] have demonstrated that the activities of intracellular enzymes, the seminal plasma protein concentrations, and the seminal plasma enzyme activities can be used to evaluate spermatozoan plasma membrane integrity. The plasma membrane of the sperm is probably the structure most susceptible to damage by water flux during cryopreservation [25]. The results reported here reveal that cryopreservation of sea bass semen did not cause injuries to spermatozoan plasma membranes; the percentage of viable cells was similar in fresh and frozen-thawed sperm, the activity of the intracellular enzymes was not decreased in frozen-thawed sperm, and the seminal plasma protein concentrations and ß-D-glucuronidase activity was not increased in frozen- thawed sperm (Table 1). These finding are supported by the observation that eosin uptake was similar in fresh and frozen-thawed sperm. Another interesting result concerns the integrity and activity of the spermatozoan mitochondria; respiration rate, malate dehydrogenase activity, and intracellular ATP concentration did not decrease in frozen- thawed spermatozoa (Table 1). The stability of ATP concentration is crucial for motility after thawing. In rainbow trout spermatozoa, ATP concentration and motility score both decreased considerably after freezing-thawing [26]. The increases in both intracellular ATP concentration and malate dehydrogenase activity that we measured in sea bass sperm after cryopreservation (Table 1) are similar to results reported for European catfish (Silurus glanis) [27]. As previously reported, the increase of intracellular ATP concentration occurs during early freezing of sperm (from +20°C to –10°C) [25]. The most probable candidate determining the increase of ATP concentration is dimethyl sulfoxide, which interferes with cellular metabolism [28].
No data are available concerning the kinetics and structure of mitochondrial malate dehydrogenase in fish. In other vertebrates, mitochondrial malate dehydrogenase shows a complex dependence on the ionic environment, which influences both kinetics and structure [29–33]. The increase of malate dehydrogenase activity after cryopreservation could be a consequence of the oxidative stress that occurs during the freezing phase, as previously suggested [11], or could be due to the presence of anions that increase the activity of the enzyme by stabilizing the dimeric form [33].
The effect of cryopreservation on osmolality and pH of seminal plasma has been evaluated; these parameters are important for sperm motility and activation in fish [34, 35]. Only pH was affected by cryopreservation of sea bass semen (Table 1).
The results obtained indicated that ATP and potassium concentrations and aspartate aminotransferase and ß-D-glucuronidase activities have a linear relationship with fertilization rate in fresh sperm. ATP concentration was positively correlated with fertilization rate (Fig. 1A); concentrations of >1.8 µmol/mg protein characterized sperm with fertilization rates 
75%. The relationship between ATP concentration and fertilization rate is due to the fact that the flagellar beat frequency of spermatozoa depends on ATP concentration and dynein ATPase activity [11, 36]. Thus, intracellular ATP concentration could be used instead of sperm motility as a predictor of fertilization ability. Determination of ATP concentration has some advantages over motility assessment: it is not subjective as is motility determination based on microscopic observation [6] and it is faster and less expensive with respect to the computer- assisted sperm analysis system.
Aspartate aminotransferase activity of sperm was negatively correlated with fertilization rate in sea bass (Fig. 1B); activities of 0.3 mU/100 mg protein characterized sperm with fertilization rates of 75%. A correlation between the activity of this enzyme and fertilization rate was also found in lake whitefish (Coregonus clupeaformis) [9] and rainbow trout [11]. The physiological meaning of this relationship is uncertain.
ß-D-Glucuronidase activity is negatively correlated with fertilization rate (Fig. 3A); activities 0.004 U/L characterized sperm with fertilization rates 75%. The enzyme ß-D-glucuronidase is involved in hydrolysis of ß-glucuronides to glucuronic acid and is located most frequently in lysosomes [37]. It is located in the spermatic duct epithelium, usually in areas where lytic processes occur, and is also secreted into the seminal fluid [38]. A correlation between this activity of this enzyme and fertilization rate was also found in rainbow trout [11]. An increase of seminal plasma ß-D-glucuronidase activity indicates degeneration or aging processes in the semen [11].
Seminal plasma potassium concentration in sea bass was negatively correlated with fertilization rate (Fig. 3B); concentrations of 17 mM potassium characterized sperm with fertilization rates of 75%. Quadratic functions were used to described the relationship between fertilization rate and potassium concentration in other fish species, i.e., bleak (Alburnus alburnus), Atlantic salmon, and rainbow trout [8, 11]. Intracellular triglyceride concentration and malate dehydrogenase activity of sea bass sperm also had a quadratic relation with fertilization rate (Fig. 2). The results obtained indicate that malate dehydrogenase activities of 0.01 U/mg protein and 0.07 U/mg protein and triglyceride concentrations of 0.14 µmol/mg protein and 0.45 µmol/mg protein characterized sperm with fertilization rates of 50%. Low malate dehydrogenase activity is indicative of poor oxidative phosphorylation, but high enzymatic activity indicates semen of low quality that has an increased energy demand [39]. A correlation between triglyceride concentration and fertilization rate was also found in rainbow trout [11]. Triglycerides are a main energetic resource for sperm, and low levels are indicative of inadequate energy resources, whereas high levels are indicative of aging processess [11, 39].
Variations in semen fertilization rate could be best described by two multiple regression models: a model including sperm parameters (ATP and aspartate aminotransferase), which accounted for 68% of the total variance in fertilization rate, and a model including seminal plasma parameters (ß-D-glucuronidase and potassium), which accounted for 66% of the total variance on fertilization rate. For practical application, the utilization of a simple regression model is valuable because the technical effort needed for parameter determination is lower. Thus, fertilization rate can be best described by one sperm parameter (ATP concentration), which accounted for 41% of the total variance in fertilization rate, and one seminal plasma parameter (ß- D-glucuronidase), which accounted for 30% of the total variance in fertilization rate. The relationship between these two parameters and fertilization rate was investigated in frozen-thawed sperm (Fig. 4) to determine semen fitness for cryopreservation and for quality control of cryopreserved semen. The results here reported indicate that quality of fresh and frozen-thawed semen can be evaluated by measurements of ATP concentration and seminal plasma ß- D-glucuronidase activity.
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FOOTNOTES |
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Received: 31 December 2003.
First decision: 9 January 2004.
Accepted: 30 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  J. Cosson, A.-L. Groison, M. Suquet, C. Fauvel, C. Dreanno, and R. Billard Marine fish spermatozoa: racing ephemeral swimmers Reproduction, September 1, 2008; 136(3): 277 - 294. [Abstract] [Full Text] [PDF]
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