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Simultaneous evaluation of superoxide content and mitochondrial membrane potential in stallion semen samples provides additional information about sperm quality
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Simultaneous evaluation of superoxide content and mitochondrial membrane potential in stallion semen samples provides additional information about sperm quality ⁎
A. Johannissona, , M.I. Figueiredoa,1, Z. Al-Kassa,b, J.M. Morrella a b
Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Box 7054, 75007 Uppsala, Sweden Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
A R T IC LE I N F O
ABS TRA CT
Keywords: Stallion Spermatozoa Cytometry Mitochondria Motility
An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoechst 33258 exclusion were measured and correlated to superoxide production and MMP. Both bivariate correlations between measured parameters as well as multivariate analysis were performed. Measured values in the superoxide/MMP assay did not correlate with other parameters. However, there was a strong negative correlation (r = 0.96 after 0 h, r = 0.95 after 24 h) between membrane integrity and chromatin integrity. Moderate positive correlations were found between motility parameters and membrane integrity, as well as moderate negative correlations between motility parameters and chromatin integrity. The multivariate analysis revealed that membrane integrity, chromatin integrity and motility contributed to the first principal component, while the second was influenced by superoxide/ MMP parameters as well as sperm kinematics. Storage of samples for 24 h decreased motility, chromatin integrity and membrane integrity. In conclusion, combined measurement of superoxide and MMP provides additional information not obtained by other assays of sperm quality.
1. Introduction Equine breeders would like to have more effective methods to analyse sperm quality in the hope of using only good-quality sperm doses for AI (Morrell et al., 2017) and thus increase pregnancy rates. The proportion of motile spermatozoa together with the number and morphology of spermatozoa in a sample is commonly used to evaluate semen quality (Varner et al., 2015). However, it is not highly correlated with the fertilizing capacity of semen samples (Graham, 1996) since motility is only one of many attributes that a spermatozoon must possess to fertilize an oocyte. A more in-depth analysis of motility can be obtained by measuring kinematics with computer-aided sperm analysis (CASA), although differences in type of counting chamber used can influence the results and act as an additional source of uncertainty (Hoogewijs et al., 2012). Measurement of sperm quality can also be analysed by metabolic activity of
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Corresponding author. E-mail address: [email protected] (A. Johannisson). 1 Present address: CECA/ICETA — Animal Sciences Centre, University of Porto, Vairao, Portugal. https://doi.org/10.1016/j.anireprosci.2018.03.030 Received 29 November 2017; Received in revised form 21 March 2018; Accepted 28 March 2018 0378-4320/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Johannisson, A., Animal Reproduction Science (2018), https://doi.org/10.1016/j.anireprosci.2018.03.030
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the spermatozoa (Gibb et al., 2014) for example, by measuring mitochondrial membrane potential (MMP, (Ortega-Ferrusola et al., 2009a)). Reactive oxygen species (ROS) are produced by all metabolising cells, thus the potential fertility of the stallion could potentially be indicated by a combination of ROS content and MMP levels contained in semen samples. Mitochondria generate a major part of the ATP required for sperm metabolism, membrane function and motility, together with anaerobic glycolysis in the cytoplasm (Peña et al., 2009). Stallion spermatozoa have a high ROS production compared with other species, since sperm ATP production comes mainly from OXPHOS (Gibb et al., 2014, Varner et al., 2015). However, in some studies (Luo et al., 2013; Gibb et al., 2014) significant correlations were found between oxidative stress parameters and a number of motility parameters, suggesting that the most fertile ejaculates were those exhibiting higher levels of ROS production. A possible explanation for the relationship between the generation of ROS and fertility might be that the most fertile sperm populations are those exhibiting the highest levels of oxidative phosphorylation (OXPHOS), with ROS as a by-product of intense mitochondrial activity (Gibb et al., 2014). The imbalance between the generation and degradation of ROS may be defined as oxidative stress (Baumber et al., 2000; Hossain et al., 2011). Under physiological conditions, ROS in low levels appear to be important for normal sperm functioning (Aitken et al., 1997) but excessive ROS-formation can affect cell viability (Aitken, 1995; Baumber et al., 2000). Hydrogen peroxide (H2O2) and superoxide (O2−) produced by spermatozoa have a functional role in Ca2+ buffering (Costello et al., 2009), apoptosis (Ortega Ferrusola et al., 2010), cell death (Peña et al., 2015), capacitation control (Agarwal et al., 2014) and sperm-oocyte fusion (Baumber et al., 2000; Aitken, 1995). Superoxide is quantitatively the predominant free radical produced by biological systems (Hybertson et al., 2011). Superoxide is short-lived and cell-impermeant (Peña et al., 2016). In the presence of nitrogen oxide (NO), superoxide can form the reactant peroxynitrite. Nitrogen oxide is more reactive than superoxide, and is produced in significant amounts by stallion spermatozoa (Ortega Ferrusola et al., 2009b). Immature, morphologically abnormal spermatozoa and seminal leukocytes are the main sources of ROS in ejaculates (Gibb et al., 2014). For many sperm preparation methods associated with assisted reproduction technology (ART), seminal plasma is removed, decreasing the antioxidant protection for spermatozoa, rendering them susceptible to oxidative stress (Baumber et al., 2000). Lower sperm quality and mitochondrial dysfunction may result in more ROS production during storage of sperm doses (Nohl et al., 1996) resulting in a negative relationship between the percentage of ROS in the sample and the foaling rate (Johannisson et al., 2014). The purpose of the present study was to investigate the relationship of a combined measurement of MMP and superoxide content to other sperm quality parameters, and to determine if this relationship changes during cold storage of semen samples. 2. Material and methods 2.1. Semen collection Commercial semen doses were obtained from 8 fertile Warmblood stallions, 4–18 years old, kept on a commercial stud in Sweden. Semen was collected up to three times per week during the breeding season; three ejaculates from six stallions and two ejaculates from two stallions were obtained in March and April. The semen was collected using an artificial vagina, Missouri model, using a phantom as a mount. Gel was removed using an in-line filter. 2.2. Sperm analysis 2.2.1. Sperm concentration The concentration of spermatozoa in raw semen was measured immediately after ejaculation using a Nucleocounter SP-100 (Chemometec, Allerød, Denmark). Subjective motility was assessed by stud personnel. Semen AI doses were prepared by adding warm (37 °C) semen extender without antibiotics (Equiplus) to a final concentration of 109 motile spermatozoa (the standard dose for cooled semen in Sweden). Antibiotics (benzyl penicillin and dihydrostreptomycin) were added to combat bacterial contamination. The extended semen was aspirated into 20-mL syringes. Immediately after collection, the extended semen doses were driven to the laboratory (1 h drive) in a Styrofoam box containing a cold pack; such packaging, maintains the temperature of semen doses at approximately 7 °C for 24 h when the ambient temperature is 20 °C (Malmgren, 1998). On arrival at the laboratory at SLU, the sperm concentration was again measured using the Nucleocounter SP-100 to establish the sperm concentration for staining the spermatozoa for flow cytometry. Following initial sperm analysis, the semen was placed in a refrigerator and the analyses were repeated after 24 h. 2.2.2. Computer-aided sperm analysis (CASA) Motility analysis (CASA) was performed using a SpermVision (Minitüb, Tiefenbach, Germany), which was connected to an Olympus BX 51 microscope (Olympus, Japan), when the samples arrived and again after 24 h. Aliquots (6 μL) of sperm samples were placed on a warm glass slide covered with an 18 × 18-mm coverslip. Motility in eight fields (∼1000 spermatozoa) was evaluated at 38 °C using the SpermVision software program. The cell identification area was set at 14–80 μm2 and spermatozoa were classified as follows: (1) immotile spermatozoa were defined as those with an average change in the orientation of the head of less than 17°; and (2) local (i.e. non-progressive) motile spermatozoa were defined as those covering a straight line distance (DSL) < 6 μm or having a circular movement with a radius < 35 μm and DSL < 30 μm. The kinematics measured were total motility (Motile), progressively motile (PMotile), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), straightness (STR), linearity (LIN), Wobble (WOB), amplitude of lateral head deviation (ALH) and beat cross frequency (BCF). 2
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2.2.3. Chromatin integrity The method used for the measurement of chromatin integrity was based on previously published methods (Johannisson et al., 2009) with minor modifications. Equal volumes (50 μL) of sperm samples and buffer containing 0.01 M Tris-HCl, 0.15 M sodium chloride and 1 mM EDTA (pH 7.4; Tris-sodium-EDTA buffer, TNE) were mixed to give a final sperm suspension of approximately 2 × 106 cells mL−1; samples were snap-frozen in liquid nitrogen before being transferred to a −80 °C freezer for storage until subsequent evaluation by flow cytometry. Samples were thawed on crushed ice immediately before staining as follows: 90 μL of TNEbuffer was added to 10 μL of each thawed sample. The TNE-extended sperm suspensions were subjected to partial DNA denaturation in situ by mixing with 0.2 m L of a low-pH detergent solution containing 0.17% Triton X-100, 0.15 M NaCl and 0.08 M HCl (pH 1.2), followed 30 s later by staining with 0.6 mL acridine orange (AO) (6 μg mL−1 in 0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, 0.15 M NaCl, pH 6.0). Measurements were made with an LSR flow cytometer (BDBiosciences, San José, CA, USA) equipped with standard optics. From each sample, a total of 10 000 events was measured at a flow rate of approximately 200 cells s−1. Green fluorescence from AO bound to double-stranded DNA was detected through a 530/30 bandpass filter, whereas red fluorescence from AO bound to single-stranded DNA was detected through a 660/20 bandpass filter. Data were collected using Cellquest version 3.3. Further calculation of DNA fragmentation index (%DFI) was performed using FCSExpress version 2 (DeNovo Software, Thornthill, Ontario, Canada).
2.2.4. Mitochondrial membrane potential and measurement of ROS Measurements of MMP were made by staining spermatozoa with the lipophilic substance 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). This dye differentially labels mitochondria according to their membrane potential, emitting in the high orange wavelength for high MMP and in the green wavelength for low MMP, when excited at 488 nm. Measurements of ROS were made by staining spermatozoa with MitoSOXRed (Invitrogen, Carlsbad, CA, USA). MitoSOXRed is a specific fluorescent probe for superoxide anion (SO%) produced by mitochondria in the cell population (Peña et al., 2016). The samples were also stained with Hoechst 33258 to exclude spermatozoa with a damaged membrane; menadione, a stimulant of ROS production, was also added. A cell suspension with 2 million sperm/mL (final volume of 300 μL) was stained. The samples were divided in two groups: the first one was incubated with a final concentration of 1.5 μM of JC-1, 3 μM of MitoSOXRed and Hoechst 33258 (1.2 μM); the second group was incubated with a final concentration of 1.5 μM of JC-1, 3 μM of MitoSOXRed, Hoechst 33258 (1.2 μM) and menadione (200 μM). After incubation for 30 min at 37 °C, the samples were analysed using a FACSVerse flow cytometer. Samples were excited with a blue laser (488 nm) and a violet laser (405 nm). Green fluorescence was detected with a bandpass filter (527/32 nm), orange fluorescence was detected using bandpass filters (586/42 nm), red fluorescence was measured using a bandpass filter (700/54 nm) and blue fluorescence was detected with a 528/45 nm bandpass filter. The data were compensated to eliminate spectral overlap. Flow Cytometry contour plots for this assay are shown in Fig. 1, without and with addition of menadione. A total of 30 000 events was evaluated and calculated as percentages of spermatozoa with high or low mitochondrial membrane potential, superoxide negative and live or superoxide positive, after gating for sperm cells in the forward scatter (FSC)-side scatter (SSC) dot-plot and also excluding spermatozoa positive for Hoechst 33258. Cells were classified as having either high or low MMP and high or low ROS production by placing a quadrant, based on the menadione-stimulated sample.
Fig. 1. Contour plots obtained by simultaneous analysis of JC-1 (x-axis) and MitoSOXRed (Y-axis). Data without addition of menadione (left) and with menadione (right) are shown. 3
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Table 1 Mean values ( ± SD) for sperm parameters after 0 and 24 h (n = 22). An asterisk indicates a significant difference, with the indicated value being the highest. Sperm Parameter
0h
24 h
P-value
Membrane Integrity (MI, %) DNA Fragmentation Index (DFI, %) Motile (%) Progressively Motile (PMotile, %) Velocity Average Path (VAP, μm/s) Velocity Curved Line (VCL, μm/s) Velocity Straight Line (VSL, μm/s) Straightness (STR, ratio) Linearity (LIN, ratio) Wobble (WOB, ratio) Amplitude of Lateral Head Displacement (ALH, μm) Beat Cross Frequency (BCF, Hz) Mitosox (MSOX)-JC+ (%) Mitosox (MSOX)+JC+ (%) Mitosox (MSOX)-JC- (%) Mitosox (MSOX) + JC- (%) Mean Fluorescence Instensity of Mitosox + (MFIMSOX) + (channel) Mean Fluorescence Instensity of Mitosox- MFIMSOX- (channel) Hoechst 33258 (HO)- (%)
63.6 ± 19.9* 29.0 ± 18.7 73.3 ± 23.3* 45.0 ± 16.8* 84.6 ± 9.2 143 ± 16 63.1 ± 7.7 0.74 ± 0.03 0.44 ± 0.03* 0.59 ± 0.03* 4.44 ± 0.55 32.8 ± 2.6* 6.48 ± 10.3 35.3 ± 25.3 9.35 ± 15.9 48.9 ± 28.8 1780 ± 1100 1450 ± 532 24.5 ± 13.7
58.1 ± 22.2 34.1 ± 21.7* 60.5 ± 26.2 35.9 ± 17.7 83.8 ± 10.6 147 ± 21 61.8 ± 8.1 0.73 ± 0.04 0.42 ± 0.05 0.57 ± 0.04 4.35 ± 0.47 31.1 ± 3.9 5.26 ± 11.5 29.2 ± 23.9 10.0 ± 9.74 55.5 ± 25.4 1850 ± 869 1740 ± 1060 22.2 ± 13.7
< 0.0001 < 0.0001 0.001 0.008 NS NS NS NS 0.024 0.017 NS 0.0496 NS NS NS NS NS NS NS
2.2.5. Membrane integrity Membrane integrity (MI) was analysed with a mixture of SYBR-14 and propidium iodide (PI; Live-Dead Sperm Viability Kit L7011; Invitrogen, Eugene, OR, USA). Aliquots with cell suspension and CellWash with 2 million sperm/mL (final volume of 300 μL) were stained with 0.6 μL SYBR-14 stock solution (diluted 1: 50 in CellWash) and 3.0 μL PI. After incubation at 37 °C for 10 minutes, spermatozoa were analysed using a FACSVerse Flow Cytometer with standard optics. A total of 30 000 events was collected and quantified as percentages of sperm populations. Samples were excited with a blue laser (488 nm). Green fluorescence was detected with an FL1 bandpass filter (527/32 nm), whereas red fluorescence was measured using an FL3 bandpass filter (700/54 nm). The spermatozoa were classified as live spermatozoa with an intact membrane (SYBR+-14/PI-), moribund (SYBR–14/PI+), and dead (SYBR+-14/PI+). 2.3. Statistical and multivariate analyses The analyses were performed using Microsoft Excel with XLSTAT addition (Addinsoft, New York, NY, USA). Comparisons between 0 and 24 h were performed using Wilcoxons signed rank test. Correlations were calculated using linear regression. For performing principal component analysis, all data were included in the modelling; separate models were used for 0 h and 24 h data. The differences were considered significant at p < 0.05. Results are presented as mean ± SD. 3. Results Mean values and significant changes between 0 and 24 h are shown in Table 1. After 24 h storage, membrane integrity decreased (P < 0.0001), the fraction of spermatozoa with unstable chromatin increased (P < 0.0001), and motility was lower (P = 0.001) compared with 0 h values. There were also changes in the kinematic parameters LIN (P = 0.024), WOB (P = 0.017) and BCF (P = 0.0496), which were all deceased by storage for 24 h. When analysing the JC-1- MitoSOXRed stained samples, it was found that in addition to an increased staining with MitoSOXRed as expected, the addition of menadione also lowered the staining for JC-1 aggregates (Fig. 1). Bivariate correlations between the different measured parameters were generally low, with the exception of a strong negative correlation between the percentage of membrane-intact spermatozoa and %DFI. This correlation had an r-value of 0.98 at 0 h and 0.97 after 24 h of cold storage (Fig. 2). There were also moderate positive correlations (0.6–0.7) between motility parameters and membrane integrity and moderate negative correlations (0.6–0.7) between motility parameters and %DFI. Total motility and progressive motility had an r-value of 0.95 (0 h) and 0.98 (24 h). Multivariate analysis by PCA revealed that while the membrane integrity, motility parameters and chromatin integrity contributed to the first principal component, the second principal component was heavily influenced by the JC-1- MitoSOXRed parameters as well as the kinetic parameters. This was true both after 0 h (Fig. 3) and 24 h (Fig. 4). The samples obtained from the same stallion mainly grouped together, indicating that major changes in sperm quality within a stallion did not occur. 4. Discussion To our knowledge, this is the first study where simultaneous measurement of JC-1 fluorescence and MitoSOXRed fluorescence has 4
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Fig. 2. Correlations between membrane integrity (X-axis) and %DFI (Y-axis) after 0 h (left) and 24 h (right).
been performed in stallion spermatozoa. The JC-1/ MitoSOXRed parameters measured in the present study did not show correlations with established markers of sperm quality such as membrane integrity, motility and chromatin integrity. However, in the multivariate analysis, the results obtained with this assay supplied explanation of the variance of results by contributing to the second principal component in the PCA-analysis. The assay may thus be valuable to include when building multiparameter models for the quality of sperm samples. The small changes between 0 h and 24 h storage indicate that the outcome of the multiparameter analysis is robust and that this analysis can be used to extract useful information from the datasets, as has been done in previous studies on bulls (Kumaresan et al., 2017). This is further corroborated by that samples from the same stallion grouped together. Such models could in the future also include the yield of spermatozoa after performing single layer centrifugation (SLC), since this yield has shown a high correlation with fertility (Morrell et al., 2014). It was somewhat surprising that the expected increase in superoxide induced by the addition of menadione was accompanied by a reduced MMP, as indicated by the lowered proportion of spermatozoa with fluorescence originating from JC-1 aggregates. However, experiments on stallion spermatozoa where another ROS-generating system, the xanthine/xanthine oxidase system, was used, indicated a similar decrease in MMP after stimulation of ROS production (Ertmer et al., 2017). We speculate that menadione initially stimulated a burst of mitochondrial activity, resulting in increased ROS production, but the membrane potential subsequently decreased, leading to dispersion of the JC-1 aggregates and decrease in orange JC-1 fluorescence. Cooling procedures damage stallion spermatozoa, as reported in the literature (Love et al., 2002) and as confirmed by our results, since we observed that the membrane integrity was lower and chromatin fragmentation higher in cooled samples. Thus, it was not surprising that the quality of the samples decayed with time. However, it is interesting to note that the correlations between measured parameters obtained in the initial samples were preserved after storage for 24 h. For the combined measurement of SO content and MMP, no difference between 0 h and 24 h could be detected, which indicates that mitochondria are robust. The lack of correlation between membrane integrity assayed by SYBR/PI staining and membrane integrity as assayed by exclusion of Hoechst 33258 was somewhat surprising, since previous studies on boar spermatozoa have shown a positive correlation > 0.99 (Huo et al., 2002). Our results showed lower percentages of membrane-intact spermatozoa when using exclusion of Hoechst 33258, which might indicate that stallion spermatozoa have less robust membranes than boar spermatozoa, and that exclusion of Hoechst 33258 as a criterion for membrane integrity should be used with caution in stallion spermatozoa. Although there was no correlation between SO% content and %DFI, %living spermatozoa, total and progressive motility, in extended, cooled and stored semen, total motility was positively correlated with membrane integrity. Motility is the most easily evaluated parameter of sperm quality, and is commonly used to decide if semen can be used for AI (Peña et al., 2015). However, it has poor predictive value for fertility (Morrell et al., 2017) especially since some motile spermatozoa may have morphologic abnormalities or damaged chromatin. Therefore, it is important to evaluate other parameters in addition to motility (Love et al., 2002). Evaluation of longevity of sperm motility in cooled samples is also important, because the transport of cooled semen with retained fertility increases the flexibility for both stallion and mare owners in selection of sires (Malmgren, 1998). When the semen of fertile stallions is stored at 5 °C, the decrease in chromatin quality is significantly lower than for subfertile stallions, where sperm DNA may have greater rate of denaturation after 20–30 h (Love et al., 2002). In horses, particularly when spermatozoa are stored or processed for later use, lipid peroxidation occurring in the plasma membrane is a major factor causing differences in sperm quality (Almeida and Ball, 2005). This damage, caused by ROS, among other factors, can alter the fluidity of the sperm membrane and the activation of signal transduction pathways, critical for sperm function 5
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Fig. 3. PCA for data obtained after 0 h. The first (X-axis) and second (Y-axis) principal components are shown. Measurement parameters indicated by dots with lines to the origin, samples by dots without connected lines. The first PC is mainly influenced by motility parameters, membrane integrity and %DFI, while the second is mainly influenced by sperm kinematics and JC-1/ MitoSOXRed parameters. Note that samples from the same stallion in most cases group together, indicating a consistency between sampling occasions.
(Storey, 2008). Chromatin integrity is one of the sperm characteristics associated with fertility. The evaluation of sperm DNA integrity is of utmost importance since early embryo development depends on the integrity of the DNA (Love and Kenney, 1998). Chromatin abnormalities and DNA damage are derived from many variables, including damage induced by ROS (Gibb et al., 2014). Stallion spermatozoa have a high ROS production compared with other species, since sperm ATP production comes majorly from OXPHOS (Gibb et al., 2014). In a study with hamster spermatozoa (Yeoman et al., 1998) hyperactivation was inhibited in presence of superoxide dismutase (SOD), indicating the potential activity of SO% as a hyperactivation stimulator. Another possibility could be that hyperactive spermatozoa, with increased mitochondrial activity, are responsible for the higher ROS production (Gibb et al., 2014). It would be interesting to investigate the relation between ROS and hyperactivity, analysing two different populations: one with spermatozoa samples incubated with menadione and another one incubated with SOD and menadione, to evaluate the percentage of hyperactive spermatozoa. In conclusion, combined measurement of superoxide and MMP provides additional information not obtained by other assays of sperm quality.
5. Declaration of interest None. 6
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Fig. 4. PCA for data obtained after 24 h The first (X-axis) and second (Y-axis) principal components are shown. Measurement parameters indicated by dots with lines to the origin, samples by dots without connected lines. The first PC is mainly influenced by motility parameters, membrane integrity and %DFI, while the second is mainly influenced by sperm kinematics and JC-1/ MitoSOXRed parameters. Note that samples from the same stallion in most cases group together, indicating a consistency between sampling occasions. Note also the similarity with results after 0 h.
Acknowledgements Stallion semen was kindly donated by Menhammar Stuteri. The work was supported by the Swedish-Norwegian Foundation for Equine Research (grant number H-14-47-008). Maria Inês Figueiredo was supported by the ERASMUS+ programme. References Agarwal, A., Virk, G., Ong, C., du Plessis, S.S., 2014. Effect of oxidative stress on male reproduction. World J. Men’s Health 32, 1–17. Aitken, R.J., 1995. Free radicals, lipid peroxidation and sperm function. Reprod. Fertil. Dev. 7, 659–668. Aitken, R.J., Fisher, H.M., Fulton, N., Gomez, E., Know, W., Lewis, B., Irvine, S., 1997. Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors iodonium and quinacrine. Mol. Reprod. Dev. 47, 468–482. Almeida, J., Ball, B.A., 2005. Effect of α-tocopherol and tocopherol succinate on lipid peroxidation in equine spermatozoa. Anim. Reprod. Sci. 87, 321–337. Baumber, J., Ball, B.A., Gravance, C.G., Medina, V., Davies-Morel, M.C., 2000. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J. Androl. 21, 895–902. Costello, S., Michelangeli, F., Nash, K., Lefievre, L., Morris, J., Machado-Oliveira, G., Barratt, C., Kirkman-Brown, J., Publicover, S., 2009. Ca2+-stores in sperm: their identities and functions. Reproduction 138, 425–437. Ertmer, F., Oldenhof, H., Schütze, S., Rohn, K., Wolkers, W.F., Sieme, H., 2017. Induced sub-lethal oxidative damage affects osmotic tolerance and cryosurvival of
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spermatozoa. Reprod. Fertil. Dev. 29, 1739–1750. Gibb, Z., Lambourne, S.R., Aitken, R.J., 2014. The paradoxical relationship between stallion fertility and oxidative stress. Biol. Reprod. 91 (77), 1–10. Graham, J.K., 1996. Analysis of stallion semen and its relation to fertility. Vet. Clin. North Am. Equine Pract. 12, 119–130. Hoogewijs, M.K., de Vliegher, S.P., Govaere, J.L., de Schauwer, C., de Kruif, A., van Soom, A., 2012. Influence of counting chamber type on CASA outcomes of equine semen analysis. Equine Vet. J. 44, 542–549. Hossain, M.S., Johannisson, A., Wallgren, M., Nagy, S., Siqueira, A.P., Rodriguez-Martinez, H., 2011. Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. Asian J. Androl. 13, 406–419. Huo, L.-J., Ma, X.-H., Yang, Z.-M., 2002. Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness in extended boar semen during long-term storage. Theriogenology 58, 1349–1360. Hybertson, R.M., Gao, B., Bose, S.K., McCord, J.M., 2011. Oxidative stress in health and disease: the therapeutic potential of Nrf2 activation. Mol. Aspects Med. 32, 234–246. Johannisson, A., Morrell, J.M., Thorén, J., Jönsson, M., Dalin, A.-M., Rodriguez-Martinez, H., 2009. Colloidal centrifugation with Androcoll-ETM prolongs stallion sperm motility, viability and chromatin integrity. Anim. Reprod. Sci. 116, 119–128. Johannisson, A., Lundgren, A., Humblot, P., Morrell, J.M., 2014. Naturally and stimulated levels of reactive oxygen species in cooled stallion semen destined for artificial insemination. Animal 8, 1706–1714. Kumaresan, A., Johannisson, A., Al-Essawe, E.M., Morrell, J.M., 2017. Sperm viability, reactive oxygen species, and DNA fragmentation index combined can discriminate between above- and below-average fertility bulls. J. Dairy Sci. 100, 5824–5836. Love, C.C., Kenney, R.M., 1998. The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. Theriogenology 50, 955–972. Love, C.C., Thompson, J.A., Lowry, V.K., Varner, D.D., 2002. Effect of storage time and temperature on stallion sperm DNA and fertility. Theriogenology 57, 1135–1142. Luo, S.-M., Schatten, H., Sun, Q.-Y., 2013. Sperm mitochondria in reproduction: good or bad and where do they go? J. Genet. Genomics 40, 549–556. Malmgren, L., 1998. Effectiveness of two systems for transporting equine semen. Theriogenology 50, 833–839. Morrell, J.M., Stuhtmann, G., Meurling, S., Lundgren, A., Winblad, C., Macias Garcia, B., Johannisson, A., 2014. Sperm yield after single layer centrifugation with Androcoll-E is related to the potential fertility of the original ejaculate. Theriogenology 81, 1005–1011. Morrell, J.M., Lagerqvist, A., Humblot, P., Johannisson, A., 2017. Effect of single layer centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen. Reprod. Fertil. Dev. 29, 1039–1045. Nohl, H., Gille, L., Schönheit, K., Liu, Y., 1996. Conditions allowing redox-cycling ubisemiquinone in mitochondria to establish a direct redox couple with molecular oxygen. Free Radical Biol. Med. 20, 207–213. Ortega-Ferrusola, C., García, B.M., Gallardo-Bolanos, J., González Fernández, L., Rodríguez Martínez, H., Tapia, J.A., Peña, F.J., 2009a. Apoptotic markers can be used to forecast the freezeability of stallion spermatozoa. Anim. Reprod. Sci. 114, 393–403. Ortega Ferrusola, C., González Fernandez, L., Macías García, B., Salazar-Sandoval, C., Morrillo Rodríguez, A., Rodríguez-Martinez, H., Tapia, J.A., Peña, F.J., 2009b. Effect of cryopreservation on nitric oxide production by stallion spermatozoa. Biol. Reprod. 81, 1106–1111. Ortega Ferrusola, C., González Fernández, L., Salazar Sandoval, C., Macías Garcia, B., Rodríguez Martínez, H., Tapia, J.A., Peña, F.J., 2010. Inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion spermatozoa. Theriogenology 74, 458–465. Peña, F.J., Rodríguez Martínez, H., Tapia, J.A., Ortega Ferrusola, C., González Fernández, L., Macias Garcia, B., 2009. Mitochondria in mammalian sperm physiology and pathology: a review. Reprod. Domest. Anim. 44, 345–349. Peña, F.J., Plaza Davila, M., Ball, B.A., Squires, E.L., Martin Muñoz, P., Ortega Ferrusola, C., Balao da Silva, C., 2015. The impact of reproductive technologies on stallion mitochondrial function. Reprod. Domest. Anim. 50, 529–537. Peña, F.J., Ortega Ferrusola, C., Martín Muñoz, P., 2016. New flow cytometry approaches in equine andrology. Theriogenology 86, 366–372. Storey, B.T., 2008. Mammalian sperm metabolism: oxygen and sugar, friend and foe. Int. J. Dev. Biol. 52, 427–437. Varner, D.D., Gibb, Z., Aitken, R.J., 2015. Stallion fertility: a focus on the spermatozoon. Equine Vet. J. 47, 16–24. Yeoman, R.R., Jones, W.D., Rizk, B.M., 1998. Evidence for nitric oxide regulation of hamster sperm hyperactivation. J. Androl. 19, 58–64.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Simultaneous evaluation of superoxide content and mitochondrial membrane potential in stallion semen samples provides additional information about sperm quality ⁎
A. Johannissona, , M.I. Figueiredoa,1, Z. Al-Kassa,b, J.M. Morrella a b
Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Box 7054, 75007 Uppsala, Sweden Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq
A R T IC LE I N F O
ABS TRA CT
Keywords: Stallion Spermatozoa Cytometry Mitochondria Motility
An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoechst 33258 exclusion were measured and correlated to superoxide production and MMP. Both bivariate correlations between measured parameters as well as multivariate analysis were performed. Measured values in the superoxide/MMP assay did not correlate with other parameters. However, there was a strong negative correlation (r = 0.96 after 0 h, r = 0.95 after 24 h) between membrane integrity and chromatin integrity. Moderate positive correlations were found between motility parameters and membrane integrity, as well as moderate negative correlations between motility parameters and chromatin integrity. The multivariate analysis revealed that membrane integrity, chromatin integrity and motility contributed to the first principal component, while the second was influenced by superoxide/ MMP parameters as well as sperm kinematics. Storage of samples for 24 h decreased motility, chromatin integrity and membrane integrity. In conclusion, combined measurement of superoxide and MMP provides additional information not obtained by other assays of sperm quality.
1. Introduction Equine breeders would like to have more effective methods to analyse sperm quality in the hope of using only good-quality sperm doses for AI (Morrell et al., 2017) and thus increase pregnancy rates. The proportion of motile spermatozoa together with the number and morphology of spermatozoa in a sample is commonly used to evaluate semen quality (Varner et al., 2015). However, it is not highly correlated with the fertilizing capacity of semen samples (Graham, 1996) since motility is only one of many attributes that a spermatozoon must possess to fertilize an oocyte. A more in-depth analysis of motility can be obtained by measuring kinematics with computer-aided sperm analysis (CASA), although differences in type of counting chamber used can influence the results and act as an additional source of uncertainty (Hoogewijs et al., 2012). Measurement of sperm quality can also be analysed by metabolic activity of
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Corresponding author. E-mail address: [email protected] (A. Johannisson). 1 Present address: CECA/ICETA — Animal Sciences Centre, University of Porto, Vairao, Portugal. https://doi.org/10.1016/j.anireprosci.2018.03.030 Received 29 November 2017; Received in revised form 21 March 2018; Accepted 28 March 2018 0378-4320/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Johannisson, A., Animal Reproduction Science (2018), https://doi.org/10.1016/j.anireprosci.2018.03.030
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the spermatozoa (Gibb et al., 2014) for example, by measuring mitochondrial membrane potential (MMP, (Ortega-Ferrusola et al., 2009a)). Reactive oxygen species (ROS) are produced by all metabolising cells, thus the potential fertility of the stallion could potentially be indicated by a combination of ROS content and MMP levels contained in semen samples. Mitochondria generate a major part of the ATP required for sperm metabolism, membrane function and motility, together with anaerobic glycolysis in the cytoplasm (Peña et al., 2009). Stallion spermatozoa have a high ROS production compared with other species, since sperm ATP production comes mainly from OXPHOS (Gibb et al., 2014, Varner et al., 2015). However, in some studies (Luo et al., 2013; Gibb et al., 2014) significant correlations were found between oxidative stress parameters and a number of motility parameters, suggesting that the most fertile ejaculates were those exhibiting higher levels of ROS production. A possible explanation for the relationship between the generation of ROS and fertility might be that the most fertile sperm populations are those exhibiting the highest levels of oxidative phosphorylation (OXPHOS), with ROS as a by-product of intense mitochondrial activity (Gibb et al., 2014). The imbalance between the generation and degradation of ROS may be defined as oxidative stress (Baumber et al., 2000; Hossain et al., 2011). Under physiological conditions, ROS in low levels appear to be important for normal sperm functioning (Aitken et al., 1997) but excessive ROS-formation can affect cell viability (Aitken, 1995; Baumber et al., 2000). Hydrogen peroxide (H2O2) and superoxide (O2−) produced by spermatozoa have a functional role in Ca2+ buffering (Costello et al., 2009), apoptosis (Ortega Ferrusola et al., 2010), cell death (Peña et al., 2015), capacitation control (Agarwal et al., 2014) and sperm-oocyte fusion (Baumber et al., 2000; Aitken, 1995). Superoxide is quantitatively the predominant free radical produced by biological systems (Hybertson et al., 2011). Superoxide is short-lived and cell-impermeant (Peña et al., 2016). In the presence of nitrogen oxide (NO), superoxide can form the reactant peroxynitrite. Nitrogen oxide is more reactive than superoxide, and is produced in significant amounts by stallion spermatozoa (Ortega Ferrusola et al., 2009b). Immature, morphologically abnormal spermatozoa and seminal leukocytes are the main sources of ROS in ejaculates (Gibb et al., 2014). For many sperm preparation methods associated with assisted reproduction technology (ART), seminal plasma is removed, decreasing the antioxidant protection for spermatozoa, rendering them susceptible to oxidative stress (Baumber et al., 2000). Lower sperm quality and mitochondrial dysfunction may result in more ROS production during storage of sperm doses (Nohl et al., 1996) resulting in a negative relationship between the percentage of ROS in the sample and the foaling rate (Johannisson et al., 2014). The purpose of the present study was to investigate the relationship of a combined measurement of MMP and superoxide content to other sperm quality parameters, and to determine if this relationship changes during cold storage of semen samples. 2. Material and methods 2.1. Semen collection Commercial semen doses were obtained from 8 fertile Warmblood stallions, 4–18 years old, kept on a commercial stud in Sweden. Semen was collected up to three times per week during the breeding season; three ejaculates from six stallions and two ejaculates from two stallions were obtained in March and April. The semen was collected using an artificial vagina, Missouri model, using a phantom as a mount. Gel was removed using an in-line filter. 2.2. Sperm analysis 2.2.1. Sperm concentration The concentration of spermatozoa in raw semen was measured immediately after ejaculation using a Nucleocounter SP-100 (Chemometec, Allerød, Denmark). Subjective motility was assessed by stud personnel. Semen AI doses were prepared by adding warm (37 °C) semen extender without antibiotics (Equiplus) to a final concentration of 109 motile spermatozoa (the standard dose for cooled semen in Sweden). Antibiotics (benzyl penicillin and dihydrostreptomycin) were added to combat bacterial contamination. The extended semen was aspirated into 20-mL syringes. Immediately after collection, the extended semen doses were driven to the laboratory (1 h drive) in a Styrofoam box containing a cold pack; such packaging, maintains the temperature of semen doses at approximately 7 °C for 24 h when the ambient temperature is 20 °C (Malmgren, 1998). On arrival at the laboratory at SLU, the sperm concentration was again measured using the Nucleocounter SP-100 to establish the sperm concentration for staining the spermatozoa for flow cytometry. Following initial sperm analysis, the semen was placed in a refrigerator and the analyses were repeated after 24 h. 2.2.2. Computer-aided sperm analysis (CASA) Motility analysis (CASA) was performed using a SpermVision (Minitüb, Tiefenbach, Germany), which was connected to an Olympus BX 51 microscope (Olympus, Japan), when the samples arrived and again after 24 h. Aliquots (6 μL) of sperm samples were placed on a warm glass slide covered with an 18 × 18-mm coverslip. Motility in eight fields (∼1000 spermatozoa) was evaluated at 38 °C using the SpermVision software program. The cell identification area was set at 14–80 μm2 and spermatozoa were classified as follows: (1) immotile spermatozoa were defined as those with an average change in the orientation of the head of less than 17°; and (2) local (i.e. non-progressive) motile spermatozoa were defined as those covering a straight line distance (DSL) < 6 μm or having a circular movement with a radius < 35 μm and DSL < 30 μm. The kinematics measured were total motility (Motile), progressively motile (PMotile), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), straightness (STR), linearity (LIN), Wobble (WOB), amplitude of lateral head deviation (ALH) and beat cross frequency (BCF). 2
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2.2.3. Chromatin integrity The method used for the measurement of chromatin integrity was based on previously published methods (Johannisson et al., 2009) with minor modifications. Equal volumes (50 μL) of sperm samples and buffer containing 0.01 M Tris-HCl, 0.15 M sodium chloride and 1 mM EDTA (pH 7.4; Tris-sodium-EDTA buffer, TNE) were mixed to give a final sperm suspension of approximately 2 × 106 cells mL−1; samples were snap-frozen in liquid nitrogen before being transferred to a −80 °C freezer for storage until subsequent evaluation by flow cytometry. Samples were thawed on crushed ice immediately before staining as follows: 90 μL of TNEbuffer was added to 10 μL of each thawed sample. The TNE-extended sperm suspensions were subjected to partial DNA denaturation in situ by mixing with 0.2 m L of a low-pH detergent solution containing 0.17% Triton X-100, 0.15 M NaCl and 0.08 M HCl (pH 1.2), followed 30 s later by staining with 0.6 mL acridine orange (AO) (6 μg mL−1 in 0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, 0.15 M NaCl, pH 6.0). Measurements were made with an LSR flow cytometer (BDBiosciences, San José, CA, USA) equipped with standard optics. From each sample, a total of 10 000 events was measured at a flow rate of approximately 200 cells s−1. Green fluorescence from AO bound to double-stranded DNA was detected through a 530/30 bandpass filter, whereas red fluorescence from AO bound to single-stranded DNA was detected through a 660/20 bandpass filter. Data were collected using Cellquest version 3.3. Further calculation of DNA fragmentation index (%DFI) was performed using FCSExpress version 2 (DeNovo Software, Thornthill, Ontario, Canada).
2.2.4. Mitochondrial membrane potential and measurement of ROS Measurements of MMP were made by staining spermatozoa with the lipophilic substance 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). This dye differentially labels mitochondria according to their membrane potential, emitting in the high orange wavelength for high MMP and in the green wavelength for low MMP, when excited at 488 nm. Measurements of ROS were made by staining spermatozoa with MitoSOXRed (Invitrogen, Carlsbad, CA, USA). MitoSOXRed is a specific fluorescent probe for superoxide anion (SO%) produced by mitochondria in the cell population (Peña et al., 2016). The samples were also stained with Hoechst 33258 to exclude spermatozoa with a damaged membrane; menadione, a stimulant of ROS production, was also added. A cell suspension with 2 million sperm/mL (final volume of 300 μL) was stained. The samples were divided in two groups: the first one was incubated with a final concentration of 1.5 μM of JC-1, 3 μM of MitoSOXRed and Hoechst 33258 (1.2 μM); the second group was incubated with a final concentration of 1.5 μM of JC-1, 3 μM of MitoSOXRed, Hoechst 33258 (1.2 μM) and menadione (200 μM). After incubation for 30 min at 37 °C, the samples were analysed using a FACSVerse flow cytometer. Samples were excited with a blue laser (488 nm) and a violet laser (405 nm). Green fluorescence was detected with a bandpass filter (527/32 nm), orange fluorescence was detected using bandpass filters (586/42 nm), red fluorescence was measured using a bandpass filter (700/54 nm) and blue fluorescence was detected with a 528/45 nm bandpass filter. The data were compensated to eliminate spectral overlap. Flow Cytometry contour plots for this assay are shown in Fig. 1, without and with addition of menadione. A total of 30 000 events was evaluated and calculated as percentages of spermatozoa with high or low mitochondrial membrane potential, superoxide negative and live or superoxide positive, after gating for sperm cells in the forward scatter (FSC)-side scatter (SSC) dot-plot and also excluding spermatozoa positive for Hoechst 33258. Cells were classified as having either high or low MMP and high or low ROS production by placing a quadrant, based on the menadione-stimulated sample.
Fig. 1. Contour plots obtained by simultaneous analysis of JC-1 (x-axis) and MitoSOXRed (Y-axis). Data without addition of menadione (left) and with menadione (right) are shown. 3
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Table 1 Mean values ( ± SD) for sperm parameters after 0 and 24 h (n = 22). An asterisk indicates a significant difference, with the indicated value being the highest. Sperm Parameter
0h
24 h
P-value
Membrane Integrity (MI, %) DNA Fragmentation Index (DFI, %) Motile (%) Progressively Motile (PMotile, %) Velocity Average Path (VAP, μm/s) Velocity Curved Line (VCL, μm/s) Velocity Straight Line (VSL, μm/s) Straightness (STR, ratio) Linearity (LIN, ratio) Wobble (WOB, ratio) Amplitude of Lateral Head Displacement (ALH, μm) Beat Cross Frequency (BCF, Hz) Mitosox (MSOX)-JC+ (%) Mitosox (MSOX)+JC+ (%) Mitosox (MSOX)-JC- (%) Mitosox (MSOX) + JC- (%) Mean Fluorescence Instensity of Mitosox + (MFIMSOX) + (channel) Mean Fluorescence Instensity of Mitosox- MFIMSOX- (channel) Hoechst 33258 (HO)- (%)
63.6 ± 19.9* 29.0 ± 18.7 73.3 ± 23.3* 45.0 ± 16.8* 84.6 ± 9.2 143 ± 16 63.1 ± 7.7 0.74 ± 0.03 0.44 ± 0.03* 0.59 ± 0.03* 4.44 ± 0.55 32.8 ± 2.6* 6.48 ± 10.3 35.3 ± 25.3 9.35 ± 15.9 48.9 ± 28.8 1780 ± 1100 1450 ± 532 24.5 ± 13.7
58.1 ± 22.2 34.1 ± 21.7* 60.5 ± 26.2 35.9 ± 17.7 83.8 ± 10.6 147 ± 21 61.8 ± 8.1 0.73 ± 0.04 0.42 ± 0.05 0.57 ± 0.04 4.35 ± 0.47 31.1 ± 3.9 5.26 ± 11.5 29.2 ± 23.9 10.0 ± 9.74 55.5 ± 25.4 1850 ± 869 1740 ± 1060 22.2 ± 13.7
< 0.0001 < 0.0001 0.001 0.008 NS NS NS NS 0.024 0.017 NS 0.0496 NS NS NS NS NS NS NS
2.2.5. Membrane integrity Membrane integrity (MI) was analysed with a mixture of SYBR-14 and propidium iodide (PI; Live-Dead Sperm Viability Kit L7011; Invitrogen, Eugene, OR, USA). Aliquots with cell suspension and CellWash with 2 million sperm/mL (final volume of 300 μL) were stained with 0.6 μL SYBR-14 stock solution (diluted 1: 50 in CellWash) and 3.0 μL PI. After incubation at 37 °C for 10 minutes, spermatozoa were analysed using a FACSVerse Flow Cytometer with standard optics. A total of 30 000 events was collected and quantified as percentages of sperm populations. Samples were excited with a blue laser (488 nm). Green fluorescence was detected with an FL1 bandpass filter (527/32 nm), whereas red fluorescence was measured using an FL3 bandpass filter (700/54 nm). The spermatozoa were classified as live spermatozoa with an intact membrane (SYBR+-14/PI-), moribund (SYBR–14/PI+), and dead (SYBR+-14/PI+). 2.3. Statistical and multivariate analyses The analyses were performed using Microsoft Excel with XLSTAT addition (Addinsoft, New York, NY, USA). Comparisons between 0 and 24 h were performed using Wilcoxons signed rank test. Correlations were calculated using linear regression. For performing principal component analysis, all data were included in the modelling; separate models were used for 0 h and 24 h data. The differences were considered significant at p < 0.05. Results are presented as mean ± SD. 3. Results Mean values and significant changes between 0 and 24 h are shown in Table 1. After 24 h storage, membrane integrity decreased (P < 0.0001), the fraction of spermatozoa with unstable chromatin increased (P < 0.0001), and motility was lower (P = 0.001) compared with 0 h values. There were also changes in the kinematic parameters LIN (P = 0.024), WOB (P = 0.017) and BCF (P = 0.0496), which were all deceased by storage for 24 h. When analysing the JC-1- MitoSOXRed stained samples, it was found that in addition to an increased staining with MitoSOXRed as expected, the addition of menadione also lowered the staining for JC-1 aggregates (Fig. 1). Bivariate correlations between the different measured parameters were generally low, with the exception of a strong negative correlation between the percentage of membrane-intact spermatozoa and %DFI. This correlation had an r-value of 0.98 at 0 h and 0.97 after 24 h of cold storage (Fig. 2). There were also moderate positive correlations (0.6–0.7) between motility parameters and membrane integrity and moderate negative correlations (0.6–0.7) between motility parameters and %DFI. Total motility and progressive motility had an r-value of 0.95 (0 h) and 0.98 (24 h). Multivariate analysis by PCA revealed that while the membrane integrity, motility parameters and chromatin integrity contributed to the first principal component, the second principal component was heavily influenced by the JC-1- MitoSOXRed parameters as well as the kinetic parameters. This was true both after 0 h (Fig. 3) and 24 h (Fig. 4). The samples obtained from the same stallion mainly grouped together, indicating that major changes in sperm quality within a stallion did not occur. 4. Discussion To our knowledge, this is the first study where simultaneous measurement of JC-1 fluorescence and MitoSOXRed fluorescence has 4
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Fig. 2. Correlations between membrane integrity (X-axis) and %DFI (Y-axis) after 0 h (left) and 24 h (right).
been performed in stallion spermatozoa. The JC-1/ MitoSOXRed parameters measured in the present study did not show correlations with established markers of sperm quality such as membrane integrity, motility and chromatin integrity. However, in the multivariate analysis, the results obtained with this assay supplied explanation of the variance of results by contributing to the second principal component in the PCA-analysis. The assay may thus be valuable to include when building multiparameter models for the quality of sperm samples. The small changes between 0 h and 24 h storage indicate that the outcome of the multiparameter analysis is robust and that this analysis can be used to extract useful information from the datasets, as has been done in previous studies on bulls (Kumaresan et al., 2017). This is further corroborated by that samples from the same stallion grouped together. Such models could in the future also include the yield of spermatozoa after performing single layer centrifugation (SLC), since this yield has shown a high correlation with fertility (Morrell et al., 2014). It was somewhat surprising that the expected increase in superoxide induced by the addition of menadione was accompanied by a reduced MMP, as indicated by the lowered proportion of spermatozoa with fluorescence originating from JC-1 aggregates. However, experiments on stallion spermatozoa where another ROS-generating system, the xanthine/xanthine oxidase system, was used, indicated a similar decrease in MMP after stimulation of ROS production (Ertmer et al., 2017). We speculate that menadione initially stimulated a burst of mitochondrial activity, resulting in increased ROS production, but the membrane potential subsequently decreased, leading to dispersion of the JC-1 aggregates and decrease in orange JC-1 fluorescence. Cooling procedures damage stallion spermatozoa, as reported in the literature (Love et al., 2002) and as confirmed by our results, since we observed that the membrane integrity was lower and chromatin fragmentation higher in cooled samples. Thus, it was not surprising that the quality of the samples decayed with time. However, it is interesting to note that the correlations between measured parameters obtained in the initial samples were preserved after storage for 24 h. For the combined measurement of SO content and MMP, no difference between 0 h and 24 h could be detected, which indicates that mitochondria are robust. The lack of correlation between membrane integrity assayed by SYBR/PI staining and membrane integrity as assayed by exclusion of Hoechst 33258 was somewhat surprising, since previous studies on boar spermatozoa have shown a positive correlation > 0.99 (Huo et al., 2002). Our results showed lower percentages of membrane-intact spermatozoa when using exclusion of Hoechst 33258, which might indicate that stallion spermatozoa have less robust membranes than boar spermatozoa, and that exclusion of Hoechst 33258 as a criterion for membrane integrity should be used with caution in stallion spermatozoa. Although there was no correlation between SO% content and %DFI, %living spermatozoa, total and progressive motility, in extended, cooled and stored semen, total motility was positively correlated with membrane integrity. Motility is the most easily evaluated parameter of sperm quality, and is commonly used to decide if semen can be used for AI (Peña et al., 2015). However, it has poor predictive value for fertility (Morrell et al., 2017) especially since some motile spermatozoa may have morphologic abnormalities or damaged chromatin. Therefore, it is important to evaluate other parameters in addition to motility (Love et al., 2002). Evaluation of longevity of sperm motility in cooled samples is also important, because the transport of cooled semen with retained fertility increases the flexibility for both stallion and mare owners in selection of sires (Malmgren, 1998). When the semen of fertile stallions is stored at 5 °C, the decrease in chromatin quality is significantly lower than for subfertile stallions, where sperm DNA may have greater rate of denaturation after 20–30 h (Love et al., 2002). In horses, particularly when spermatozoa are stored or processed for later use, lipid peroxidation occurring in the plasma membrane is a major factor causing differences in sperm quality (Almeida and Ball, 2005). This damage, caused by ROS, among other factors, can alter the fluidity of the sperm membrane and the activation of signal transduction pathways, critical for sperm function 5
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Fig. 3. PCA for data obtained after 0 h. The first (X-axis) and second (Y-axis) principal components are shown. Measurement parameters indicated by dots with lines to the origin, samples by dots without connected lines. The first PC is mainly influenced by motility parameters, membrane integrity and %DFI, while the second is mainly influenced by sperm kinematics and JC-1/ MitoSOXRed parameters. Note that samples from the same stallion in most cases group together, indicating a consistency between sampling occasions.
(Storey, 2008). Chromatin integrity is one of the sperm characteristics associated with fertility. The evaluation of sperm DNA integrity is of utmost importance since early embryo development depends on the integrity of the DNA (Love and Kenney, 1998). Chromatin abnormalities and DNA damage are derived from many variables, including damage induced by ROS (Gibb et al., 2014). Stallion spermatozoa have a high ROS production compared with other species, since sperm ATP production comes majorly from OXPHOS (Gibb et al., 2014). In a study with hamster spermatozoa (Yeoman et al., 1998) hyperactivation was inhibited in presence of superoxide dismutase (SOD), indicating the potential activity of SO% as a hyperactivation stimulator. Another possibility could be that hyperactive spermatozoa, with increased mitochondrial activity, are responsible for the higher ROS production (Gibb et al., 2014). It would be interesting to investigate the relation between ROS and hyperactivity, analysing two different populations: one with spermatozoa samples incubated with menadione and another one incubated with SOD and menadione, to evaluate the percentage of hyperactive spermatozoa. In conclusion, combined measurement of superoxide and MMP provides additional information not obtained by other assays of sperm quality.
5. Declaration of interest None. 6
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Fig. 4. PCA for data obtained after 24 h The first (X-axis) and second (Y-axis) principal components are shown. Measurement parameters indicated by dots with lines to the origin, samples by dots without connected lines. The first PC is mainly influenced by motility parameters, membrane integrity and %DFI, while the second is mainly influenced by sperm kinematics and JC-1/ MitoSOXRed parameters. Note that samples from the same stallion in most cases group together, indicating a consistency between sampling occasions. Note also the similarity with results after 0 h.
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