Dagmar Waberski, Unit for Reproductive Medicine of Clinics, University of Veterinary Medicine Hannover, Bünteweg 15, D-30559 Hannover, Germany. E-mail: firstname.lastname@example.org
Boar spermatozoa are sensitive to storage temperatures below 15 °C. Chilling injury causes loss of motility and membrane integrity in a minority of cells, whereas the main population displays sublethal changes compromising fertility. In this study, changes of the response to capacitation conditions in hypothermically stored boar spermatozoa have been examined using a kinetic approach with well-defined test and control media. Ejaculates of seven boars were diluted in Beltsville Thawing Solution kept for 3 h at 22 °C or cooled to 17, 10 and 5 °C and stored for 24 and 96 h. At each time point, the standard sperm parameters motility and membrane integrity were evaluated. Subsequently, washed subsamples were incubated in capacitating and control medium before flow cytometric analysis of intracellular calcium content using the Fluo-3 probe and changes in phospholipid disorder using merocyanine. Kinetic changes of response parameters were monitored in viable (plasma membrane intact) cells. Chilling led to a loss of standard sperm quality traits in a minor subpopulation of cells, whereas storage length had no effect on these parameters. However, responses to incubation as determined by the loss of live cells with low intracellular calcium content showed marked changes in relation to storage conditions. The specific responsiveness to capacitation conditions decreased in close relation to storage temperature and length. In contrast, the merocyanine probe revealed to be limited to detect effects of hypothermic storage. Using Fourier transform infrared spectroscopy, no influence of chilling on membrane phase behaviour was found that might implicate decreased sperm function. In conclusion, assessment of response to capacitating media by monitoring intracellular calcium levels provides a sensitive measure for chilling injury in extended boar semen, and therefore, deserves implementation in hypothermic storage tests.
Among mammalian species, boar spermatozoa are especially sensitive towards cold shock and, therefore, provide an excellent model to study the response of spermatozoa to chilling and hypothermic storage. Commonly, boar spermatozoa are stored in liquid form at 15–17 °C for routine use in artificial insemination. The boar spermatozoa's sensitivity to chilling below 15 °C has been attributed to the lipid composition of their plasma membranes, which have a relatively high content of polyunsaturated fatty acids and a low sterol to phospholipid ratio (Parks & Lynch, 1992). Damage during cooling in the suprazero temperature range is referred to as chilling injury, the extent of which depends on the chilling rate and some threshold temperature (Mazur et al., 2008). The most obvious signs of chilling and storage injury to a subpopulation of spermatozoa are an irreversible loss of motility and loss of membrane integrity (reviewed in: Watson & Plummer, 1985), both attributes of lethal cell damage. There is evidence, however, that the major, surviving population of spermatozoa within a semen sample shows impaired cell function resulting in disturbed sperm transport, altered sperm-oviduct interaction and shortened survival in the female reproductive tract (Watson, 1995). Such sperm damage commonly remains undetected using conventional semen analysis, for example, motility and viability (Waberski et al., 2011). The plasma membrane appears to be the primary site of chilling injury. Cooling of cells from physiological temperatures down to temperatures just above the freezing point induces phase separation and rearrangement of membrane components, as has been shown in freeze-fracture studies (Holt & North, 1984; De Leeuw et al., 1990). Fourier transform infrared spectroscopy (FTIR) studies have indicated that cold shock damage is associated with passage through membrane phase transitions (Drobnis et al., 1993; Arav et al., 2000). It has been suggested that the rearrangement of lipid domains during cooling and re-warming results in an increase in membrane permeability for solutes including ions, such as calcium (Green & Watson, 2001). Uptake of external calcium has been associated with ongoing destabilization of spermatozoa, which partially resembles the capacitation process and thereby presumably compromises fertility (Bailey & Buhr, 1995; Green & Watson, 2001). It has been shown that spermatozoa with high intracellular calcium levels display lower ability to bind to the oviductal epithelium and altered capacitation behaviour in vitro (Dobrinski et al., 1996; Petrunkina et al., 2001). Of particular interest is that differences in response to cooling are seen, both between cells and ejaculates (Holt et al., 2005; Petrunkina et al., 2005a). There is evidence from previous studies using kinetic single cell analysis that the cytosolic calcium content in boar spermatozoa progresses as a function of time during incubation under capacitating conditions in populations of responding spermatozoa (Harrison et al., 1993; Petrunkina et al., 2005b). Cooling-induced membrane destabilization seems somehow to accelerate dynamics of acrosome reaction and cell death in response to capacitation conditions (Petrunkina et al., 2005a). In contrast, other studies report that boar spermatozoa cooled and re-warmed in the presence of bicarbonate exhibited a lesser membrane response compared with fresh spermatozoa incubated at 39 °C (Green & Watson, 2001; Guthrie & Welch, 2005a). So far, sensitive measures for chilling-associated loss of sperm function in extended boar semen are lacking in spermatology laboratories. The responsiveness as assessed by changes in intracellular calcium levels has been shown to detect detrimental effects of storage in liquid preserved boar spermatozoa (Petrunkina et al., 2005a); however, a close relationship to storage temperature yet has not been established.
The aim of the present study was to investigate the effect of chilling and subsequent long-term storage of extended boar semen on specific responses to capacitation stimuli as monitored by intracellular calcium levels and phospholipid disorder. Our hypothesis was that changes in this response could prove to be a more sensitive indicator of chilling-associated loss of sperm function compared with motility and viability parameters. Moreover, FTIR was used to determine the temperature range at which boar spermatozoa conventionally stored at 17 °C exhibit thermotropic phase transitions, and to examine whether membrane phase behaviour was affected by storage at hypothermic temperatures.
Materials and methods
Unless otherwise stated, chemicals were obtained from Merck AG (Darmstadt, Germany), Roth GmbH (Karlsruhe, Germany) and Sigma-Aldrich (Steinheim, Germany). Percoll was purchased from GE Healthcare (Munich, Germany). Yo-Pro-1 was obtained from Molecular Probes (Eugene, OR, USA). Fluo-3-AM and fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA) were purchased from Axxora GmbH (Lörrach, Germany). Gentamicin sulfate was purchased from Serva Electrophoresis GmbH (Heidelberg, Germany). BTS extender was obtained from Minitüb GmbH (Tiefenbach, Germany).
Semen collection and dilution
Semen was collected from seven boars (4 Pietrain, 1 Duroc-crossbreed, 1 Hampshire-crossbreed, 1 Large White, ages between 2 and 8 years) held at the facilities of the Unit for Reproductive Medicine of Clinics at the University of Veterinary Medicine Hannover. Ejaculates were collected by the gloved hand method into disposable filtering bags (Minitüb GmbH) that were enclosed in insulated plastic thermos cups pre-heated to 38 °C. Immediately after collection, semen was transferred to the laboratory and isothermically (33 °C) diluted with BTS (Beltsville Thawing Solution; Johnson et al., 1988) to a final concentration of 2 × 107 cells/mL. After dilution, samples of 100 mL were kept at 22 °C room temperature for 3 h, after which they were analysed. At this time point, samples had reached 22 °C. Alternatively, samples were cooled down to 17, 10 or 5 °C using the following cooling regime: After 90 min holding time at 22 °C, samples were transferred to a 17 °C cooling chamber. Samples to be stored at 10 °C were held at 17 °C for 60 min. prior to cooling to 10 °C. Samples to be stored at 5 °C were held for additional 60 min at 10 °C before they were transferred to 5 °C. This resulted in a cooling rate of approximately 0.1 °C/min. Samples were stored up to 96 h.
Semen storage and time points of semen evaluation
Semen samples were first analysed at 3 h after dilution and holding at 22 °C. Further analyses were performed in diluted semen samples stored at 17, 10 or 5 °C at two time points of storage, that is, 24 and 96 h. Conventional sperm parameters (motility, membrane integrity) were assessed as described below. At the same time points of holding and storage at the various temperatures, diluted semen subsamples were washed through a Percoll gradient, loaded with fluorescent dyes and incubated for 120 min in Tyrode's media for the determination of the intracellular calcium content and the stainability with merocyanine 540 as described below. Evaluations were performed at several time points during incubation in Tyrode's media to obtain kinetic profiles.
Conventional sperm parameters
Sperm motility was evaluated subjectively for native ejaculates within 15 min after collection using phase contrast microscopy. Sperm morphology was similarly evaluated according to Krause (1966), semen was fixated in formol citrate (Hancock, 1956) and 200 spermatozoa cells were evaluated using phase contrast microscopy. Only ejaculates with greater than 70% motility and less than 30% morphologically abnormal spermatozoa were used for experiments. A total number of seven ejaculates (one from each boar) were used.
Sperm motility during the experiments was assessed using computer-assisted sperm analysis (CASA-system Sperm Vision®; Minitüb GmbH, Tiefenbach, Germany). After warming the samples for 15 min at 38 °C in a water bath, a counting chamber of 20 μm depth (Leja Products B.V., Nieuw-Vennep, the Netherlands) was loaded with 2.7 μL sample. Spermatozoa were observed using a 20× negative-phase contrast objective, a 10× ocular and a 0.75× adaptor. Analysis was based on examination of 30 consecutive digitalized images obtained from a single field. Ten image fields and at least 400 spermatozoa per sample were analysed. The following motility parameters were evaluated: total motility (in %), VSL (straight-line velocity, in μm/sec), LIN (VSL/VCL; linearity coefficient, relative unit), ALH (amplitude of lateral head displacement, in μm) and BCF (beat cross frequency, in Hz).
Plasma membrane integrity was assessed using the cell-impermeable fluorescent dye propidium iodide (PI). Fluorescein-isothiocyanate conjugated peanut agglutinin (FITC-PNA) was used to stain the cells with a damaged acrosomal membrane, as it specifically binds to the inner leaflet of the outer acrosomal membrane (Flesch et al., 1998). Flow cytometric analysis of PI/FITC-PNA stained samples was performed on a DAKO Galaxy flow cytometer (Dako Cytomation GmbH, Hamburg, Germany), equipped with a 488 nm blue argon laser and BP 537.5/22.5, BP 590/25 and LP 630 nm filters for green (FITC), orange and red (PI) fluorescence respectively. A Hepes-buffered saline medium (137 mm NaCl, 10 mm glucose, 2.5 mm KOH, 20 mm Hepes, 300 mosmol, pH 7.40 at 38 °C) was pre-warmed to 38 °C and used as sheath fluid. The forward against side light scatter plot (both in linear mode) was used to select sperm events, and fluorescence intensities (in logarithmic mode) were collected for 10 000 events per sample, at a rate of 400–800 events/sec. Data were corrected for non-DNA containing particles as proposed by Petrunkina et al. (2010). Percentage of spermatozoa with intact plasma (PI-neg.) and acrosomal (FITC-PNA-neg.) membranes was determined and compensated for the overlap of emission spectra of PI and FITC-PNA using FloMax® software (Partec GmbH, Münster, Germany).
Incubation under capacitating conditions and in control medium
Two types of a Tyrode medium were used for exposing spermatozoa to capacitating or non-capacitating conditions (Harrison et al., 1996). Tyrode capacitating medium consisted of 96 mm NaCl, 3.1 mm KCl, 0.4 mm MgSO4, 5 mm glucose, 15 mm NaHCO3, 2 mm CaCl2, 0.3 mm KH2PO4, 20 mm Hepes, 21.6 mm sodium lactate, 1 mm sodium pyruvate, 3 mg BSA/mL, 100 μg gentamicin/mL and 20 μg phenol red/mL (pH 7.4 under 5% CO2 at 38 °C, 300 mOsmol/kg). In non-capacitating (control) medium NaHCO3 and CaCl2 were omitted, and 1 mm Na-EGTA was added. In addition, the NaCl content was increased to 112 mm to obtain a final osmolality of 300 mOsmol/kg and the pH was adjusted to be 7.4 at 38 °C using NaOH.
Prior to dilution in Tyrode capacitation or control medium, spermatozoa were separated from extender by Percoll centrifugation according to Harrison et al. (1993). Aliquots of 4 mL extended semen stored at various temperatures were carefully layered on a discontinuous Percoll-gradient (35 and 70%) and washed by centrifugation at 22 °C for 10 min at 300 × g, followed by 15 min at 750 × g without stopping the centrifuge. After centrifugation, the supernatant layers were removed leaving about 0.8 mL of the 70% Percoll solution, in which the loosely pelleted spermatozoa were gently resuspended.
Intracellular calcium content
For determining the intracellular calcium content, spermatozoa were first loaded with Fluo-3. Fluo-3-AM (4 μL in DMSO) was added to 0.8 mL Percoll-washed spermatozoa. After a 10 min incubation, this solution was diluted with saline medium (137 mm NaCl, 10 mm glucose, 2.5 mm KOH, 20 mm Hepes, 0.5 mg/mL PVA, and 0.5 mg/mL PVP, buffered to pH 7.40 with NaOH) to a final volume of 4 mL. To remove extracellular Fluo-3-AM, samples were then washed by two consecutive centrifugations (10 min at 300 × g, followed by 15 min at 750 × g without stopping the centrifuge) through a sucrose washing medium (232 mm sucrose, 10 mm glucose, 20 mm Hepes, 2.5 mm KOH, 0.5 mg/mL PVA, and 0.5 mg/mL PVP) (Harrison et al., 1993).
Aliquots of 5 μL sucrose-washed spermatozoa were diluted in 995 μL of capacitating Tyrode or control medium supplemented with 3.7 μm PI, resulting in 1–1.5 × 105 cells/mL. Spermatozoa in control medium were incubated at 38 °C in airtight closed tubes, whereas spermatozoa in Tyrode capacitation medium were incubated at 38 °C under humidified atmosphere containing 5% CO2. Samples were analysed after 3, 20, 40, 60, 90 and 120 min using the flow cytometer described above and detecting Fluo-3 fluorescence using the green filter. The sperm population was selected in the forward against side light scatter plot by their characteristic distribution patterns and agglutinated events were excluded from further analysis. As spermatozoa samples were washed through Percoll, there was no need to correct data for non-sperm events (Petrunkina & Harrison, 2010).
Flow cytometric analysis of responsiveness focused on evaluation of response in live cells specifically, rather than total populations, separately from the general monitoring of cell death (cf. Petrunkina & Harrison, 2011). Therefore, percentages of live cells with low intracellular calcium content (PI-neg./Fluo-3-neg.) and live cells with high calcium content (PI-neg./Fluo-3-pos.) as well as percentages of PI-positive cells were determined and plotted as a function of the incubation time. To describe the change in a sperm subpopulation because of incubation conditions, the responsiveness was determined. A representative time point during incubation in Tyrode was chosen and the responsiveness was calculated as the difference between the percentage of cells at the chosen time point and at the onset of incubation (TyrodeΔ60-3) in the three sperm populations. This was also done for the three sperm populations in the control medium (controlΔ60-3). The specific response to the capacitating stimuli (bicarbonate/calcium) was calculated as the difference between the responsiveness in the low-calcium live cells in capacitating Tyrode′s and control medium.
Changes of membrane phospholipid disorder in response to exposure to capacitating conditions were studied using a Yo-Pro-1/M540 double staining, as previously described by Harrison et al. (1996). Percoll-washed spermatozoa were diluted in Tyrode or control medium supplemented with 25 nm Yo-Pro-1 (YP) to enable discrimination between plasma membrane intact and damaged cells. Incubations were done as described above, at 38 °C in the presence and absence of CO2 for 2 h. Merocyanine 540 (M540) was added at a final concentration of 2.7 μm for 2 min prior to analysis. Samples were analysed at 2, 10, 20, 30, 40, 60, 90 and 120 min by flow cytometry as described above, using the green filter to detect YP and the orange filter for M540. Percentages of live cells with a basic (YP-neg./M540-neg.) or an increased (YP-neg./M540-pos.) state of phospholipid disorder as well as of plasma membrane-defective (YP-pos.) cells were determined and plotted as a function of the incubation time.
Infrared spectra were recorded on a Perkin–Elmer 100 Fourier transform infrared (FTIR) spectrometer (Perkin–Elmer, Norwalk, CT, USA), equipped with a narrow band Mercury/Cadmium/Telluride liquid nitrogen cooled IR-detector, as described previously (Oldenhof et al., 2010). Diluted semen was centrifuged for 30 sec at 14 000 g to obtain a pellet of about 60 × 107 cells. Approximately 10 μL of the pellet were mounted between two CaF2 windows and placed into a temperature-controlled sample holder. The sample was cooled from 38 to 0 °C at 2 °C/min, after which it was heated to 80 °C at 2 °C/min. Spectra were collected every 20 sec during cooling and warming.
Spectral analysis was carried out as described in detail by Wolkers & Oldenhof (2010). Membrane fluidity was monitored by observing the band position of the CH2 symmetric stretching vibration around 2 850 cm−1. For band position analysis, the spectral region between 3 000 and 2 800 cm−1 was selected, the inverted second derivative spectrum was calculated, and the lipid band was selected and normalized. Band positions were calculated by taking the average of the spectral positions at 80% of the peak height. The band position of the CH2 stretching band was plotted as a function of temperature to determine membrane phase changes.
Data were analysed using sas software (Version 9.1; sas Inst. Inc., Cary, NC, USA). A multifactorial analysis of variance (PROC GLM) was performed to determine the influence of storage temperature and storage time, and the interaction between the two factors. Data were tested for normal distribution (PROC UNIVARIATE). Comparisons between storage temperatures, storage times and incubation media were performed using Wilcoxon's signed rank or student's t-tests for repeated measurements (PROC UNIVARIATE). At each storage temperature and time, comparisons between incubation times within sperm populations were performed using Wilcoxon's signed rank or student's t-tests for repeated measurements (PROC UNIVARIATE). Data are shown as means ± standard deviation. Differences were considered to be significant, if the probability of their occurring by chance was less than 5% (p < 0.05).
Membrane integrity and motility parameters
There was a significant interaction (p < 0.05) between storage temperature and storage time. Both influenced motility and membrane integrity. Membrane integrity and motility parameters of boar spermatozoa stored at various temperatures are shown in Table 1. Plasma and acrosomal membrane integrity as well as total motility decreased significantly (p < 0.05) with decreasing storage temperature. Storage for 24 h at 17 °C resulted in a moderate decline in viability of only 2%, whereas storage at 10 or 5 °C resulted in a much greater decrease of 10 and 23% respectively. For all storage temperatures tested, storage up to 96 h did not result in further changes in membrane integrity and total motility as compared with 24 h storage. The percentage of motile spermatozoa decreased (p < 0.05) from 90% for samples stored at room temperature or 17 °C to below 70% for samples stored at 5 °C for 24 h. In addition to total motility, the kinetic parameters velocity (VSL) and linearity (LIN) decreased (p < 0.05) with decreasing temperature. For samples stored at 17 and 10 °C, respectively, other parameters did not change with increased storage time. For spermatozoa stored at 5 °C linearity decreased and head movement (ALH) increased (p < 0.05) upon storage for 96 h as compared with 24 h.
Table 1. Membrane integrity and motility parameters of boar spermatozoa stored at various temperatures
VSL: straight-line velocity; LIN: linearity; ALH: amplitude of lateral head displacement; BCF: beat cross frequency.
Data from single ejaculates from seven different boars are presented as means ± standard deviation.
Different letters within a row indicate significant differences between storage conditions (p < 0.05).
Membrane intact [%]
86.0 ± 3.4a
84.4 ± 2.5b
76.7 ± 4.6c
63.3 ± 13.7d
83.5 ± 2.3b
78.0 ± 4.1c
63.8 ± 11.9d
Total motility [%]
91.9 ± 1.8a
89.9 ± 2.6b
83.7 ± 2.9c
69.3 ± 10.1d
86.4 ± 4.4b,c
82.9 ± 5.8c
63.7 ± 16.8d
54.6 ± 10.2a,b
55.4 ± 6.5a
51.6 ± 8.3a,c
47.0 ± 7.9c,d
51.4 ± 5.6a'c'd
48.5 ± 5.4b,c,d
45.8 ± 4.7d
0.59 ± 0.0a
0.55 ± 0.04a
0.52 ± 0.06b
0.48 ± 0.06c
0.57 ± 0.09a,b
0.54 ± 0.08a,b,c
0.43 ± 0.09d
2.1 ± 0.3a,b
2.0 ± 0.3a
2.0 ± 0.3a
1.9 ± 0.3a
1.9 ± 0.5a
2.2 ± 0.5b
39.8 ± 3.0a,b
40.7 ± 2.4a
40.0 ± 3.3a,b
39.5 ± 4.0a,b
40.0 ± 1.9a,b
39.2 ± 2.6b
39.4 ± 3.0b
Changes in intracellular calcium content
Storage temperature had a significant influence (p < 0.001) on intracellular calcium content, whereas storage time did not a reveal an influence. Figure 1 shows kinetic changes in sperm subpopulations during incubation in Tyrode or control medium after dilution of freshly collected semen samples and chilling to 22 °C. Capacitation-associated changes in intracellular calcium content of live spermatozoa are presented as changes in percentages of live cells with basal and increased Fluo-3-fluorescence as a function of time. The percentage of high-calcium live spermatozoa (PI-neg./Fluo-3-pos.) increased significantly (p < 0.001) during the first 40 min of incubation in Tyrode, after which the level did not change further. In control medium, no increase in this subpopulation of spermatozoa was seen and percentages remained at 1.7–3.1%. In capacitating Tyrode, low-calcium live spermatozoa (PI-neg./Fluo-3-neg.) decreased (p < 0.0001) from around 90% at the onset of incubation to approximately 16% after 2 h incubation, whereas only a small decrease (p < 0.01) down to about 80% was observed in control medium. In capacitating Tyrode medium, the number of plasma membrane damaged (PI-positive) cells increased (p < 0.0001) from 8.5 ± 0.9% to 65.9 ± 8.4%. In contrast, in control medium a linear increase (p < 0.01) with incubation time was observed, from 6.7 ± 1.8% to only 17.3 ± 2.9%.
Figure 2 shows the percentages of total destabilized (sum of high-calcium live and PI-pos.) cells after 3 min exposure to Tyrode (part A) and control medium (part B). During 24 h of storage, the amount of destabilized cells in the Tyrode medium increased significantly with decreasing storage temperature from 12.9 ± 1.3% at 22 °C and 14.4 ± 4.2% at 17 °C to 17.4 ± 5.0% at 10 °C or 25.4 ± 7.3% at 5°C (p < 0.05). In control medium there was also a significant (p < 0.05) effect of storage temperature on the amount of destabilized cells when comparing 22 °C (8.4 ± 1.7%) with 10 °C (11.8 ± 2.8%) and 5 °C (16.4 ± 5.5%), respectively. In both media this increase was because of a significant (p < 0.05) increase in the high-calcium live cells, whereas the amount of PI-positive cells did not differ (p > 0.05).
The responsiveness, calculated as the difference between the percentage of cells at 60 and 3 min of incubation in capacitating or control medium, was used to describe the change in a sperm subpopulation owing to incubation conditions (Fig. 3A and B). Regarding the population of live spermatozoa with basic intracellular calcium content (PI-neg./Fluo-3-neg.), in semen samples hold for 3 h at 22 °C the responsiveness (∆3-60) in Tyrode (part A) was 63.6 ± 7.4% and declined gradually with decreasing temperatures in samples stored for 24 h to 47.0 ± 9.9% at 5 °C (p < 0.05). In contrast, in the control medium, the responsiveness based on this population increased significantly (p < 0.05) from 6.3 ± 3.5% at 22 °C to 22.6 ± 8.6% at 5 °C (p < 0.05; part B). In Tyrode, the responsiveness (∆60-3) in high-calcium live spermatozoa (PI-neg./Fluo-3-pos.) dropped from 16.8 ± 8.4% in freshly diluted semen hold for 3 h at 22 °C to 4.3 ± 9.0% in semen stored at 5 °C for 24 h (p < 0.05; part A). On the other hand, in control medium the responsiveness increased from 0.6 ± 1.3% in semen group 3 h/22 °C to 3.4 ± 6.9% after storage at 5 °C (p < 0.05; part B). In the capacitating medium, the responsiveness of cells with a defective plasma membrane (PI-pos. cells) did not differ between the semen storage groups (∆60-3, part A). In contrast, in the control medium PI-pos. spermatozoa increased significantly (p < 0.05) when semen was stored for 24 h at 10 °C (11.7 ± 3.4%) or 5 °C (19.1 ± 7.5%) compared with semen stored at 17 °C (part B).
The specific response to the capacitating stimuli was calculated as the difference between the responsiveness (∆3-60) in the low-calcium live cells in Tyrode and control medium (Fig. 4). During the first 24 h of storage, the specific response dropped with temperature from 57.3 ± 7.3% at 22 °C to 24.4 ± 14.9% for samples stored at 5 °C (p < 0.05). As storage was prolonged to 96 h, it decreased significantly (p < 0.05) for all temperatures tested. Samples stored at 10 °C or 5 °C for 96 h did not differ in specific response.
Changes in phospholipid disorder
To determine changes in membrane phospholipid disorder of live cells, a Yo-Pro-1/M540 double staining was performed. Storage temperature had a significant influence (p < 0.001) on phospholipid disorder, whereas storage time did not reveal an influence. Figure 5 shows percentages of live cells with basal and increased phospholipid disorder as a function of time under capacitating as well as non-capacitating conditions, for semen that was diluted in BTS and cooled down to 22 °C. The amount of merocyanine-positive live (YP-neg./M540-pos.) spermatozoa rapidly increased (p < 0.001) during the first 20 min of incubation in Tyrode medium, after which the level did not further change. In control medium, no increase in the proportion of cells with a higher state of phospholipid disorder was observed and percentages remained at 2.6–4.5%. In capacitating Tyrode medium, merocyanine-negative live (YP-neg./M540-neg.) spermatozoa decreased from around 90% at the onset of incubation to approximately 20% after 120 min (p < 0.0001), whereas a decrease down to only about 80% (p < 0.05) was observed in control medium. In capacitating Tyrode medium, the number of plasma membrane damaged (YP-pos.) cells increased (p < 0.0001) from 8.0 ± 1.8% to 58.7 ± 10.3%. In contrast, in control medium a linear increase with incubation from 7.7 ± 1.7% to only 18.7 ± 3.2% was observed.
In vitro capacitation properties of boar spermatozoa stored at various temperatures were compared. The percentage of merocyanine-positive live spermatozoa (YP-neg./M540-pos.) was determined after 2 and 20 min incubation in (non-)capacitating conditions (Table 2). After 2 min incubation, this population was significantly increased (p < 0.05) in samples stored for 24 h at 17 °C (5.4 ± 1.3%) or 5 °C (11.5 ± 5.5%), as compared with samples that were stored at 22 °C (3.7 ± 1.6%) before incubation in capacitating Tyrode medium. Also in control medium an increase (p < 0.05) in YP-neg./M540-pos. cells took place, which could be observed in samples stored for 24 h at 10 °C or 5 °C as compared with 17 or 22 °C. In contrast, these differences diminished for spermatozoa stored at different temperatures after 20 min incubation in Tyrode or control medium (p > 0.05).
Table 2. Percentages of live cells with an increased state of phospholipid disorder (YP-neg./M540-pos.), for spermatozoa exposed to capacitating (Tyrode) or non-capacitating (control) media for 2 and 20 min. Semen was diluted in BTS and kept at room temperature for 3 h or stored at 17 °C, 10 °C or 5 °C for 24 h before incubation in either medium
Data are means ± standard deviation from single ejaculates from seven different boars.
Differences that are statistically different in a row (p < 0.05) are indicated with different letters.
3.7 ± 1.6a
5.4 ± 1.3b
5.7 ± 2.9a,b
11.5 ± 5.5c
25.2 ± 10.5a
22.2 ± 6.4a
22.8 ± 5.3a
21.4 ± 3.1a
2.6 ± 0.8a
2.6 ± 0.7a
4.5 ± 1.6b
5.6 ± 3.1b
3.3 ± 0.8a,b
3.5 ± 0.9a
3.7 ± 1.9a,b
7.1 ± 2.7b
The responsiveness, calculated as the difference between the percentage of cells at 20 and 2 min of incubation in capacitating or control medium, was used to describe the change in a sperm subpopulation because of incubation conditions (Fig. 6A and B). Regarding the population of live spermatozoa with basic phospholipid disorder (YP-neg./M540-neg.), there was no significant difference in responsiveness (∆20-2) between semen samples stored under various conditions before incubation in Tyrode (part A) or control (part B) medium (p > 0.05). In Tyrode, the responsiveness (∆20-2) in live sperm with increased state of phospholipid disorder (YP-neg./M540-pos.) dropped from 21.5 ± 10.7% in freshly diluted semen hold for 3 h at 22 °C to 9.9 ± 9.8% in semen stored at 5 °C for 24 h (p < 0.05; part A); there was no significant difference for semen stored at 17 and 10 °C compared with fresh semen hold at 22 °C. In control medium, the responsiveness was between 0.6 ± 1.0% and 1.5 ± 2.7% for all storage conditions (p > 0.05). In both media, the responsiveness of cells with a defective plasma membrane (YP-pos. cells) did not differ between the semen storage groups (∆20-2, parts A and B).
Thermotropic membrane phase behaviour
To determine whether different storage temperatures and periods affect the thermotropic membrane phase behaviour of boar spermatozoa, FTIR was used. Figure 7 shows a plot of the band position of the symmetric CH2 stretching vibration around 2 850 cm−1 (νCH2) arising from the lipid acyl chains as a function of the sample temperature. The wavenumber position of this band provides a measure for membrane conformational disorder or membrane fluidity. Membrane fluidity increases with increasing temperature, with a discontinuity from this linear increase in the temperature range from about 10–30 °C, indicating a membrane phase transition or reorganization of membrane components. No differences in this characteristic thermotropic phase behaviour were observed amongst samples that were stored at different temperatures.
As expected, conventional traits of sperm quality such as CASA motility and membrane integrity revealed only small changes in extended boar semen stored below 17 °C up to 96 h. Earlier studies indicated that sensitive detection of stress-induced changes in sperm function is more promising when assays test for differences in temporal or quantitative responses under conditions that support in vitro fertilization (Holt & Van Look, 2004; Petrunkina et al., 2007; Rodriguez-Martinez & Barth, 2007). The ability of spermatozoa to undergo capacitation in a time-dependent manner has been recognized as a crucial parameter of sperm function (Harrison et al., 1993; Petrunkina et al., 2005b). The sperm population within a semen sample is highly heterogeneous and, therefore, individual cells display individual reactions to stress caused from dilution, chilling and storage. Consequently, it can be assumed that sensitive detection of distinct sperm responsiveness parameters in cell subpopulations is advantageous to identify temperature and storage effects in diluted semen samples. Following the suggestions of Harrison et al. (1993) and Petrunkina et al. (2007), in this study a kinetic experimental approach with well-defined test and control media was used, considering equally both initial response and subsequent kinetics.
We demonstrated that flow cytometric monitoring of cytosolic calcium levels in response to capacitation conditions reveals major chilling-related changes in hypothermically stored boar semen. The decrease in specific response to capacitating stimuli was closely related to storage temperature. Most noticeable, this functional response parameter proved to be markedly more sensitive than computer-assisted analysis of motility and flow cytometric assessment of membrane integrity.
As expected from original studies (Harrison et al., 1993), incubation under capacitating conditions induced dramatic changes in membrane permeability in the majority of spermatozoa even in freshly extended semen. Fluo-3 detection of capacitation-mediated changes reached plateau values between 40 and 60 min of incubation time. An influence of storage conditions on membrane response parameters was already detectable at the beginning of incubation under capacitating conditions. The proportion of spermatozoa with disturbed membrane permeability as assessed by high intracellular calcium levels and stainability with propidium iodide differed significantly between storage temperatures being highest in semen cooled to 5 °C. This indicates that even moderate, step-wise chilling induces membrane leakage or modified specific ion channel activity in a subpopulation of sensitive, rapidly destabilizing spermatozoa, similar to that described for rapid cooling (Simpson & White, 1986; Robertson et al., 1988; Bailey & Buhr, 1995). Interestingly, an increase in cytosolic calcium was also found at the onset of incubation in the bicarbonate- and calcium-free control medium supplied with chelating EGTA (see Fig. 2B). This suggests that calcium release from internal stores (reviewed by Costello et al., 2009) contributes to the loss of calcium homeostasis by chilling-induced malfunction of store-regulating ion pumps.
Detecting subtle dynamics of tested parameters requires essential maintenance of membrane integrity and reactivity below threshold values in a defined time-frame (Petrunkina et al., 2005a). Capacitation has to be regarded as a transient stage of progressive membrane destabilization, which small sperm cohorts pass in individual time-frames. Thus, at a given time point only low numbers of live, responding cells are detectable. In view of this, Harrison et al. (1993) proposed to focus on changes in the population of low-calcium live spermatozoa through kinetic approaches. Therefore, we used this sperm population to assess the specific responsiveness to capacitating conditions by calculating the difference between the responses to capacitating and control media within 60 min of incubation. Spermatozoa stored at 5 °C for 24 h displayed a lower specific responsiveness (24.4%) compared with spermatozoa 3 h after dilution hold at room temperature (57.3%) and spermatozoa stored for 24 h at 17 °C (46.7%) and 10 °C (36.1%) respectively. Chilling – and to less extent storage – reduces the specific response of spermatozoa to capacitating conditions in two different ways: First, as storage temperature was lowered, there was an overall decrease of responsiveness in low-calcium live cells which was not accompanied by an increasing destabilization rate in sperm. Second, chilling resulted in an ongoing destabilization of spermatozoa during incubation per se (i.e. in the non-capacitating control medium, see Fig. 3B). In a similar way, two opposing effects of storage, namely that part of the sperm population becomes refractory towards capacitation stimuli whereas another part becomes increasingly unstable during storage, have been shown recently for boar spermatozoa stored long-term at 17 °C (Henning et al., 2012). This study demonstrates that chilling provokes changes in spermatozoa membrane stability and capacitation dynamics, and that the extent of these changes was more strongly related to chilling temperature than to length of storage. Notably, loss of specific response to capacitating conditions was already detectable after 24 h in semen cooled to 17 °C indicating that even short-term storage at a temperature routinely used in porcine artificial insemination affects membrane function in a subpopulation of cells.
Although the molecular pathways of responses to capacitating stimuli in boar spermatozoa are now well-established (recently reviewed by Fraser, 2010 and Visconti et al., 2011), the underlying reason for chilling-induced alteration of calcium regulation is yet largely unknown. An accumulation of intracellular calcium has been reported during liquid hypothermic storage and after cryopreservation in various species (White, 1993; Mc Laughin & Frod, 1994; Kim et al., 2008). Current concepts attribute elevated cytosolic calcium to increased entry through the cell membrane and release of this ion from intracellular stores. Recently it was found that sperm freezing impairs calcium signalling: cryopreserved boar spermatozoa exposed to elevated external potassium show a smaller increase of calcium compared with fresh spermatozoa (Kim et al., 2008). The authors suggest that the degree of calcium influx through voltage-dependent calcium channel may decrease after cryopreservation. Moreover, cryopreserved human and boar spermatozoa show reduced progesterone-induced calcium signals presumably because of modification of progesterone receptors or depolarization-dependent calcium influx pathways (Mc Laughin & Frod, 1994; Rossato et al., 2000; Kim et al., 2008). The described reduced responsiveness of cryopreserved spermatozoa as detected by rise of cytosolic calcium targets the role of calcium in the acrosome reaction process. It remains speculative whether similar effects on calcium channels and membrane receptors are involved in the impaired response to capacitating signals observed in this study.
Increase in calcium occurs further downstream in the capacitation process compared with early events which are detectable within seconds after exposure to capacitation stimuli. Cyclic AMP rises to a maximum within 60 sec, and the increase in PKA-dependent protein phosphorylation begins within 90 sec (Harrison & Miller, 2000; Harrison, 2004). Changes in phospholipid asymmetry can be detected within minutes by merocyanine 540, an impermeant lipophilic probe which binds to plasma membranes with increasing affinity as their lipid components become more disordered. In boar spermatozoa, this event is independent of the presence of extracellular calcium (Harrison et al., 1996). Such changes in plasma membrane architecture could possibly alter steady-state intracellular calcium concentration, either by increasing the intrinsic permeability of the membrane (‘leakage’) or by modifying specific ion channel activity (Harrison & Gadella, 2005). There is evidence from a number of studies including our own that cooling and storage of boar spermatozoa does not induce major basal changes in membrane fluidity as detected by merocyanine (Green & Watson, 2001; Waterhouse et al., 2004; Guthrie & Welch, 2005a; Martín-Hidalgo et al., 2011). However, it was found that under capacitation conditions, the percentage of spermatozoa with high membrane lipid disorder within the viable sperm population is reduced in cooled and cryopreserved (and re-warmed) semen compared with fresh semen (Green & Watson, 2001; Guthrie & Welch, 2005a,b). We hypothesized, therefore, that kinetics of collapse of phospholipid asymmetry determined by the M540 probe would be changed because of chilling similar to those observed for intracellular calcium and this precedes changes in calcium kinetics. As expected, a small population of spermatozoa showed a rapid collapse in membrane asymmetry after about 2 min of incubation in capacitation media. Similar to the differences in initial intracellular calcium levels, a significantly higher population of spermatozoa with increased phospholipid disorder (live, M540-positive cells) was found in samples stored at 5 °C compared with 10 and 17 °C respectively. In line with previous studies using the M540 in cooled and cryopreserved boar spermatozoa (Green & Watson, 2001; Guthrie & Welch, 2005a,b), this supports the concept that chilling induces membrane lipid changes determined as ‘capacitation-like’ events rendering a subpopulation of spermatozoa more sensitive to capacitation stimuli. However, although kinetics of merocyanine stainability seem to be similar to those of intracellular calcium levels, at later incubation times when merocyanine-positive cells had reached maximal values, differences related to storage temperature or length were no longer detectable. Thus, the sensitivity of the M540 to detect chilling-associated membrane changes seems to be limited.
Passage through phase transitions has been correlated with leakage of solutes because of co-existence of domains of different membrane components and phase separation (Drobnis et al., 1993). We used FTIR to assess the temperature range in which spermatozoa pass through membrane phase transitions. Each lipid has its characteristic melting temperature, and biological membranes exhibit a membrane phase behaviour dependent on the composition of their membranes. Alterations in lipid composition that can occur upon storage affect the membrane phase behaviour; for example, FTIR revealed multiple sharp membrane phase transitions in erythrocytes after 5 days storage at 4 °C which were associated with changes in lipid composition (Wolkers et al., 2002). In contrast, our study demonstrates that chilling to 5 °C and storage at this temperature for up to 96 h does not affect the membrane phase behaviour profiles compared with boar spermatozoa stored at the conventionally used temperature of 17 °C. These findings may indicate that hypothermic storage conditions have no drastic effects on the lipid composition and organization of the cellular membranes. However, the FTIR method only reveals the phase change temperature range of the total heterogeneous lipids in all the spermatozoa cell membranes, and changes in specific membranes or in sperm subpopulations with innate population-specific differences in membrane fluidity between storage temperatures could have remained undetected. The sensitivity of this method with respect to detecting different storage temperature effects on cells with multiple membrane compartments warrants further investigation.
In conclusion, hypothermic storage resulted in a decrease in the specific responsiveness to capacitating stimuli in extended boar spermatozoa, which was closely related to chilling temperature, and to less extent, to the storage length. Loss of calcium homeostasis proved to be a more sensitive parameter of chilling-associated injury in hypothermically stored boar spermatozoa compared with conventional sperm quality traits. Chilling impairs the specific responsiveness to capacitating stimuli in two ways: (i) fast destabilization of spermatozoa membranes leading to transiently elevated intracellular calcium and subsequently to cell death, and (ii) formation of an increasingly refractory (non-responding) cell population. The merocyanine probe 540 seems to be limited for detection of chilling-related membrane destabilization. Decreased membrane function in hypothermically stored semen was not associated with changes in thermotropic membrane phase behaviour. The underlying mechanisms by which chilling disturbs calcium homeostasis of boar spermatozoa under capacitating conditions remain to be elucidated. Overall, the specific responsiveness to capacitation stimuli as monitored by changes of intracellular calcium levels provides a sensitive tool to assess new liquid preservation strategies of hypothermically stored semen. The presented post-chilling responsiveness test may be valuable for AI industry to select boar semen for hypothermic liquid storage or cryopreservation.
This work was supported by the Association for Biotechnology Research (FBF e.V., Bonn, Germany). Willem Wolkers was financially supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG), Cluster of Excellence ‘From regenerative biology to reconstructive therapy’ (REBIRTH).
Conflict of interest
The authors declare that no competing interests exist.
Performed the research: SS, HH, HO; Designed the research study: DW, HH, AP, HO, WW; Contributed essential elements or tools: DW, WW; Analysed the data. SS, HH, AP, HO; Wrote the manuscript: SS, DW, HO, AP, WW.