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Mitochondrial membrane potential and reactive oxygen species in liquid stored and cryopreserved turkey (Meleagris gallopavo) spermatozoa
Mitochondrial membrane potential and reactive oxygen species in liquid stored and cryopreserved turkey (Meleagris gallopavo) spermatozoa1 M. Slowi´ nska,2 E. Liszewska, S. Judycka, M. Konopka, and A. Ciereszko
ABSTRACT The extensive use of artificial insemination in turkeys has led to the development of in vitro semen storage. However, fertility rates from liquid stored and frozen/thawed turkey semen are still unsatisfactory. The aim of the study was to assess spermatozoa viability, mitochondrial membrane potential (MMP), and reactive oxygen species production (ROS) in liquid stored and cryopreserved turkey semen with the use of flow cytometry. Moreover, motility and adenosine triphosphate (ATP) content in sperm were monitored at the same time to link flow cytometry data with sperm movement and energetics. Liquid storage led to a decrease in sperm motility (80.6 vs. 55.6%, for fresh and stored for 48 h), live sperm with an intact MMP (59.9 vs. 30.5% for fresh and stored for 48 h), and a 20-fold decrease in ATP content after 24 h of storage. A 3-fold increase in ROS+ sperm was observed after 48 h of storage (9.3 vs. 26.8% for fresh and stored for 48 h). Semen equili-
bration before cryopreservation affected only ATP content. However, freezing/thawing led to a dramatic decrease in all of the studied semen quality parameters. A 5-fold decrease in live sperm with intact MMP (59.8 vs. 11.9%) and a 7-fold increase in sperm ROS+ (10.8 vs. 74.4%) were recorded between fresh and frozen/thawed semen. The results strongly suggested that a significant loss of MMP and a disturbance in sperm ATP production during semen storage can be the main reason for the decline in sperm motility. The disturbance of mitochondria activity during storage seems to be associated with the increase in oxidative stress in turkey semen. Turkey sperm mitochondria also appear to be very sensitive to cryodamage. Diminished energy production in turkey spermatozoa, visible as the low percentage of sperm with an intact MMP and low level of ATP after freezing/thawing, which is associated with high ROS generation, could be responsible for the low fertilizing ability of cryopreserved turkey semen.
Key words: flow cytometry, semen, liquid storage, cryopreservation, turkey 2018 Poultry Science 97:3709–3717 http://dx.doi.org/10.3382/ps/pey209
INTRODUCTION The extensive use of artificial insemination in turkeys has led to the development of in vitro semen storage in a liquid form (Thurston, 1995; Blesbois and Brillard, 2007). The liquid storage of semen is of practical interest in the management of male turkeys. However, turkey semen cannot be stored for longer than 6 h without a loss of fertilizing ability, even if oxygenated and stored with appropriate diluents at a reduced temperature (Sexton and Giesen, 1983; Thurston, 1995). Turkey semen storage is accompanied by an irreversible reduction in motility, morphological integrity, and fertility C 2018 Poultry Science Association Inc. Received October 11, 2017. Accepted April 27, 2018. 1 This work was supported by the National Science Centre, Poland (2016/21/B/NZ9/03583) and funds appropriated to the Institute of Animal Reproduction and Food Research. 2 Corresponding author: Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research Polish Academy of Sciences Tuwima 10, 10-748 Olsztyn, Poland Tel.: +48 895393135; Fax: +48 895357421; E-mail: [email protected]
of spermatozoa (Clarke et al., 1982; Thurston, 1995; Donoghue and Donoghue, 1997). Many factors may contribute to the loss of fertilizing ability of spermatozoa stored in vitro, and factors affecting lipid biochemistry, including significant losses in the phospholipids of the plasma membrane, lipid peroxidation, and greater susceptibility to damage of cold-stored sperm membranes seem to be predominant (Donoghue et al., 1996; Douard et al., 2000, 2005; Breque et al., 2003; Long and Kramer, 2003). However, the mechanisms for sperm quality loss during liquid storage are currently unknown. Similarly to liquid storage, the poultry industry has not been able to take advantage of cryopreservation (Blesbois, 2007). The unique physiological features make avian spermatozoa highly sensitive to damage caused by cryopreservation (Long, 2006). Firstly, the elongated head with small cytoplasm content causes the less effective uptake of cryoprotectant by the cell (Donoghue and Wishart, 2000). Secondly, the avian sperm tail is approximately 8 times the length of the sperm head, which also predisposes poultry sperm to sensitivity to freezing damage (Donoghue and
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Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Department of Gamete and Embryo Biology, Tuwima 10, 10-747 Olsztyn, Poland
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MATERIALS AND METHODS Birds, Housing, and Management Turkey BUT Big-6 line (British United Turkeys, Limited, Chester, England) was maintained under standard husbandry conditions at the Turkey Testing Farm in Frednowy, Poland. Males were photostimulated at 26 wk of age (14 L:10 D) and produced semen by 30 wk of age. Feed and water were available ad libitum. Individual semen samples designated for short-term storage and cryopreservation were collected by abdominal massage (Burrows and Quinns, 1937) during the routine collection of semen for artificial insemination.
Semen Storage Liquid storage Immediately after semen collection, individual semen samples were diluted (1:2) in Poultry Semen Extender (Ovodyl, IMV Technologies, I’Aigle, France). Samples of extended semen were stored for 48 h under refrigeration (4 to 7◦ C). Semen samples (n = 9) were shaken in aerobic conditions with an agitator (JW Electronic, Warsaw, Poland). Aliquots were removed from each sample 2.5, 24, and 48 h after semen collection for the measurements of semen quality parameters. At one time, no more than 3 semen samples were analyzed and every week 3 consecutive samples were collected and stored for analysis. Cryopreservation The cryopreservation procedure was performed according to the method developed by Iaffaldano et al. (2011) with some modification. Immediately after semen collection, individual semen samples were diluted (1:4) in Ovodyl. Samples of extended semen (n = 13) were cooled at 4◦ C for 60 min and then dimethylacetamide was added as a cryoprotectant to a final concentration of 8%. The diluted semen was gently inverted and equilibrated at 4◦ C for 5 min. Volumes of 80 μL of semen were plunged drop by drop directly into liquid nitrogen. The pellets were thawed by placing 1 pellet into a plastic 2.0 mL cryogenic tube and incubated in a water bath at 80◦ C for 60 s. Aliquots were removed from fresh semen, after the addition of dimethylacetamide (equilibration) and after freezing/thawing for the measurements of semen quality parameters. At one time, no more than 3 semen samples were collected for experiments and 3 consecutive samples were collected for further analysis each week.
Semen quality analysis Computer-Assisted Sperm Analysis Video recordings of motile sperm after 2.5, 24, and 48 h of liquid storage and in fresh, equilibrated and frozen/thawed semen were made using a microscope with a ×10 negative phase objective and a Sony CCD black and white video camera (SPT-M108CE). Video recordings were analyzed using Hobson Sperm Tracker (Hobson Vision Ltd, Baslow UK). MicroCell 2-chamber (20-μm depth of the chamber) slides (Conception Technologies, San Diego, CA) were used. The slides were mounted on a heated stage (41◦ C). Conditions of sperm dilutions, recordings, and Hobson Tracker settings followed those described by King et al. (2000) and Kotlowska et al. (2007) for turkey spermatozoa. Semen samples were incubated for 5 min in the motility buffer (50 mM Tris buffer, pH 7.4, 120 mM NaCl, 10 mM glucose, 2 mM CaCl2 ) at 39◦ C. Turkey semen was diluted 300 to 500 times to obtain 30 to 50 sperm on the screen at the start of motility. The motility buffer was supplemented with 0.5% BSA to prevent the adherence of spermatozoa to the glass slides. Flow Cytometry Flow cytometry analyses were performed using a Muse Cell Analyzer (EMD Millipore,
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Wishart, 2000; Long, 2006). Damage occurring during freeze-thawing first concerns the plasma membrane, mitochondria, and acrosome region and the nuclei (Blesbois, 2007). Such damage affects fertility and the duration of fertility, which is a very important factor in birds due to the long-term semen storage in the uterovaginal glands of the female (Blesbois and Brillard, 2007). Extending the knowledge concerning sperm cryoinjuries is a prerequisite for a better understanding of damage to sperm and the improvement of cryopreservation protocols for turkey semen. Recently, flow cytometry has gained popularity for avian semen quality analysis (Donoghue et al., 1995; Blanco et al., 2000; Christensen et al., 2004). Using flow cytometry, the large number of spermatozoa (>10 0000) is analyzed in a small volume of sample in a short time with the use of fluorescent probe allowing for observation of physical characteristics such as cell size, shape, and also any component or function of the spermatozoa. To date, the flow cytometry was applied for the comprehensive analysis of fresh and frozen-thawed fowl semen quality (Partyka et al., 2010; 2011a,b, 2012a). Plasma membrane integrity, acrosome integrity, mitochondrial function, and chromatin status, as well as lipid peroxidation, were assessed in fresh and cryopreserved semen of rooster and goose (Partyka et al., 2011a,b). However, only restricted analyses, regarding the measurements of sperm viability, membrane integrity, and the carbohydrate content of sperm glycocalyx, have been performed in turkey semen (Donoghue et al., 1995; Donoghue and Donoghue, 1997; Pel´ aez and Long, 2007; Long and Conn, 2012). Taking into account that fertility rates from liquid stored and frozen/thawed turkey semen have been consistently lower than that from stored chicken semen, there is a need for more detailed flow cytometry analyses of turkey semen. The aim of the present study was to assess spermatozoa viability, mitochondrial membrane potential (MMP), and reactive oxygen species production (ROS) in liquid stored and cryopreserved turkey semen with the use of flow cytometry. Moreover, motility and adenosine triphosphate (ATP) content in sperm were monitored at the same time to link flow cytometry data with sperm movement and energetics.
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performed, containing only sperm with a depolarized membrane. The positive control was performed by incubating the sperm cells in the presence of 50 μmol/L carbonyl cyanide m-chlorophenylhydrazone (SigmaAldrich, St. Louis, MO) for 15 min at 37◦ C according to Marchetti et al. (2002). ATP Concentration ATP concentration in turkey spermatozoa was measured using the ATP Colorimetric/Fluorometric Assay Kit (Sigma-Aldrich). Samples were prepared according to the method of Long and Guthrie (2006). Sample preparation included phosphatase inhibitor cocktail 2 (Sigma-Aldrich) treatment, ATP extraction (boiling water bath, 10 min), and ATP assay. ATP concentration was determined by phosphorylating glycerol, resulting in a fluorometric (λex = 535/λem = 587 nm) product proportional to the amount of ATP present.
Statistical Analysis The results are presented as mean ± SD. All analyses were performed at a significance level of P < 0.05 using GraphPad Prism software v.6.02 (GraphPad Software Inc. San Diego, CA). A 1-way ANOVA, followed by the Tukey test for post hoc comparison of means and Student’s t test, was used for analysis. For statistical procedures, the percentage data were normalized by arcsine square root transformation. Correlations between sperm quality parameters during liquid storage and cryopreservation were calculated using the Spearman correlation test.
RESULTS Motility and Motility Characteristics A steady decline in the percentage of sperm motility was observed during the storage; a significant decrease occurred after 48 h. No changes in other sperm motility characteristics related to both speed of movement (VCL, VSL and VAP) and trajectory of movement (BCF, ALH, LIN) were found at the same time (Table 1). Equilibration did not cause decrease in sperm motility and sperm motility characteristics (Table 1). A 4-fold decrease in the percentage of sperm motility was found in frozen/thawed semen. Among all sperm motility characteristics, only VSL was decreased after freezing/thawing (Table 1).
Viability No changes in sperm viability were found during 48 h of storage (Figure 1A, Supplementary Figure S1). The sperm viability was maintained at a high level (approximately 93%) for up to 48 h of storage. Equilibration did not affect sperm viability (Figure 1A’). The sperm viability in fresh and equilibrated
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Billerica, USA). Viability was assessed using Muse Count and Viability Reagent (EMD Millipore) according to the manufacturer’s protocol. A membranepermeant DNA-staining dye stained all cells with a nucleus, which is then used to discriminate cells with a nucleus from debris and non-nucleated cells. A DNAbinding Muse dye based on 7-aminoactinomycin D (7-AAD) stains cells that have lost their membrane integrity, allowing the dye to stain the nucleus of dead and dying cells. This parameter was displayed as viability and was used to discriminate viable (live cells that do not stain) from non-viable (dead or dying cells that stain) cells. Semen was diluted in PBS to obtain concentrations in the range of 1 × 105 to 1 × 107 sperm/mL. Subsequently, 20 μL of each semen suspension was mixed with 380 μL of Muse Count and Viability Reagent and incubated for 5 min at room temperature. The ROS production of spermatozoa was determined using a Muse Oxidative Stress Kit (EMD Millipore) according to the manufacturer’s protocol. Semen was diluted in 1X Assay Buffer to obtain concentrations in the range of 1 × 106 to 1 × 107 sperm/mL and then 10 μL of semen suspension was mixed with 190 μL of Muse Oxidative Stress working solution and incubated at 37◦ C for 30 min. The Muse Oxidative Stress Reagent is based on dihydroethidium (DHE), a well-characterized reagent that has been used extensively to detect ROS in cellular populations (Bindokas et al., 1996). Upon reaction with superoxide anions, DHE undergoes oxidation to form the DNA-binding fluorophore ethidium bromide or a structurally similar product which intercalates with DNA, resulting in red fluorescence. Two populations of cells were distinguished in the assay: ROS–, live cells, and ROS+, cells exhibiting ROS. To accurately determine the dividing point between ROS– and ROS+ populations, a positive control setup was performed, containing completely oxidized sperm cells. The positive control involved incubating sperm cells in the presence of 3% H2 O2 , a superoxide anion generator, for 1 h at 37◦ C. The MMP was determined using Muse MitoPotential Kit (EDM Millipore) according to the manufacturer’s protocol. Semen was diluted in 1X Assay Buffer to obtain a concentration in the range of 1 × 104 to 5 × 105 sperm/mL and then 100 μL of semen suspension was mixed with 95 μL of Muse MitoPotential working solution and incubated at 37◦ C for 20 min. Next, 5 μL of Muse 7-AAD was added to the sample and incubated at room temperature for 5 min. The Muse MitoPotential Reagent is based on MitoPotential Dye, a cationic, lipophilic dye, which detects changes in the MMP, and 7-AAD as an indicator of cell death. Four populations of cells were distinguished in the assay: live cells with depolarized MMP; live cells with intact MMP; dead cells with depolarized MMP; and dead cells with intact MMP. To accurately determine the dividing point between live cells with intact and depolarized mitochondrial membranes, a positive control setup was
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Table 1. Effect of time of liquid storage and cryopreservation on turkey spermatozoa motility and motility characteristics. Computer-assisted sperm analysis Liquid storage
Cryopreservation Motility (%) VCL (μ m/s) VSL (μ m/s) VAP (μ m/s) LIN (%) BCF (Hz) ALH (μ m)
80.6 153.5 84.5 120.4 53.3 10.7 8.0
Time of storage (h) 24 5.1a 71.6 ± 13.9a 55.6 10.2 150.5 ± 8.6 151.3 15.7 94.4 ± 12.9 98.0 15.6 122.1 ± 10.9 123.8 6.7 60.7 ± 6.9 63.1 1.3 11.5 ± 1.3 11.3 1.3 6.9 ± 1.8 6.4
Fresh 78.1 141.0 81.6 105.0 52.9 11.2 6.5
± ± ± ± ± ± ±
48 ± ± ± ± ± ± ±
7.3b 7.5 12.2 9.2 8.2 1.8 1.2
Equilibrated Frozen/thawed
10.6 74.8 ± 15.5a 19.6 ± 10.0b 16.4 143.8 ± 18.4 130.9 ± 13.2 21.5a,b 89.2 ± 24.2a 66.2 ± 26.0b 20.7 109.9 ± 22.7 95.4 ± 21.0 10.3 57.3 ± 12.0 47.1 ± 15.1 2.0 12.58 ± 2.7 10.33 ± 2.9 1.6 6.0 ± 1.2 6.9 ± 1.7 a
Data represent mean values ± SD a,b Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation. VSL = straight-line velocity; VCL = curvilinear velocity; VAP = average path velocity; LIN = linearity; BCF = beat-cross frequency; ALH = amplitude of lateral head displacement.
semen was maintained at a high level (about 90%). However, a 4-fold decrease in sperm viability was found after freezing/thawing (Figure 1A’, Supplementary Figure S1).
Mitochondrial Membrane Potential Two populations of live sperm were distinguished in fresh semen: live sperm with an intact MMP (approximately 60%) and live sperm with a depolarized MMP (approximately 30%). Storage significantly reduced the percentage of live sperm with an intact MMP at 24 and 48 h (Figure 2A). On the other hand, an increase in the percentage of live sperm with a depolarized MMP was observed after 24 and 48 h of storage (Figure 2B, Supplementary Figure S2). No changes in depolar-
Reactive Oxygen Species Fresh semen was characterized by the presence of sperm cells exhibiting ROS (approximately 10%). An increase in the percentage of sperm undergoing oxidative stress by 2- and 2.5-fold was found after 24 and 48 h of storage, respectively (Figure 3A, Supplementary Figure S3). Equilibration did not affect the percentage of ROS+ sperm. An almost 7-fold increase in the percentage of sperm undergoing oxidative stress was observed after freezing/thawing (Figure 3A’, Supplementary Figure S3).
ATP Content An almost 20-fold decrease in the ATP content was found after 24 h of storage (Figure 4A). A 2- and 40-fold decreases in ATP content were observed for equilibrated and frozen/thawed sperm, respectively (Figure 4A’).
Correlations between Sperm Quality Parameters Correlations between liquid stored sperm quality parameters are presented in Table 2. Motility, viability, life sperm with intact MMP, and ATP content were negatively correlated with ROS+. Live sperm with an intact
Figure 1. Effect of time of liquid storage (A) and cryopreservation (A’) on sperm viability. Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
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Motility (%) VCL (μ m/s) VSL (μ m/s) VAP (μ m/s) LIN (%) BCF (Hz) ALH (μ m)
2 ± ± ± ± ± ± ±
ized/dead sperm were observed (Figure 2C, Supplementary Figure S2). Equilibration did not affect the MMP (Figure 2A’and B’). A decreased percentage of both live sperm with intact MMP (5-fold, Figure 2A’) and sperm with depolarized MMP was observed after freezing/thawing (Figure 2B’). With the decrease of live sperm (with intact and depolarized mitochondrial membrane), a 9-fold increase of depolarized/dead sperm was observed (Figure 2C’, Supplementary Figure S2).
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MMP were positively correlated with motility and ATP content. Correlations between cryopreserved sperm quality parameters are presented in Table 2. Motility, viability, live sperm with an intact MMP, and ATP content were negatively correlated with ROS+. Viability, live sperm with an intact MMP, and ATP content were positively correlated with motility. Viability and live sperm with intact mitochondrial membrane were positively correlated with ATP content.
DISCUSSION In our study, a steady decline in the percentage of sperm motility was observed during storage; however, a significant decrease occurred after 48 h of storage. Those results are in agreement with previously published studies. The decline in semen motility is usually observed after 24 h (Douard et al., 2000, 2004; Slowi´ nska et al., 2012, 2013) or 48 h of storage with decrease to 40% of sperm motility (Douard et al., 2000, 2004; Iaffaldano et al., 2005; Kotlowska et al., 2007;
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Figure 2. Effect of time of liquid storage (A, B, C) and cryopreservation (A’, B’, C’) on live sperm with intact (A, A’), depolarized mitochondrial membrane (B, B’), and dead sperm with depolarized mitochondrial membrane (C, C’). Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
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Figure 4. Effect of time of liquid storage (A) and cryopreservation (A’) on sperm ATP concentration. Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
Zaniboni and Cerolini, 2009). Contrary to liquid storage, a dramatic effect on sperm motility was observed after cryopreservation. Approximately 20% of sperm motility was obtained after freezing/thawing in our study, which is in the range of 15 to 60% of sperm motility obtained after the cryopreservation of turkey semen (Iaffaldano et al., 2011, 2016; Lemoine et al., 2011; Slowi´ nska et al., 2012; Long et al. 2014). The application of computer-assisted sperm analysis for monitoring sperm motility allowed us to confirm that the procedure of liquid semen storage and cryopreservation produce a different range of decline in the percentage of sperm motility. The decline in sperm motility observed in our study during storage correlated (r = 0.38∗ ) with the decrease in MMP, measured for the first time for turkey semen. It is known that sperm motility is primarily dependent on mitochondrial function (Auger et al., 1989; Folgerø et al., 1993). Mitochondria, located in the sperm midpiece, generate a major part of the ATP required for sperm motility (Jamieson, 2006; Agnihotri et al., 2016). A significant loss of the MMP and a disturbance in sperm ATP production during semen storage, as observed in our study, can be the indicators for semen
quality. Therefore, it can be suggested that a disturbance to energy metabolism can be the reason of decline in sperm motility during turkey semen storage. It has to be mentioned that a decrease in mitochondrial potential after 24 h of storage appeared to be more pronounced (decrease by 33%) than a decrease in sperm motility (decrease by 11%). For this reason, MMP can be a potential marker of semen quality during storage. This suggestion is supported by the ATP decrease during liquid storage observed in our study and the studies of Douard et al. (2004) and Long and Guthrie (2006). It is known that poultry sperm mitochondria are sensitive to the freeze/thaw process and therefore ATP production is seriously compromised after cryopreservation (Long, 2006). In our study, we observed that turkey sperm mitochondria were also affected by cryopreservation. Firstly, we observed a 5-fold decrease in the population of live sperm with intact MMP and secondly the 40-fold decrease of ATP content of sperm after cryopreservation. A decrease in energy production in turkey spermatozoa, visible in a low percentage of sperm with an intact MMP and a low level of ATP after freezing/thawing, could be responsible for the low fertilizing ability of cryopreserved turkey semen. Our
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Figure 3. Effect of time of liquid storage (A) and cryopreservation (A’) on ROS+ sperm. Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
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FLOW CYTOMETRY OF TURKEY SEMEN Table 2. Correlations between liquid stored and cryopreserved sperm characteristics of turkey semen.
Motility (%) ATP (nmol/109 sperm) ROS+ (%) Live sperm with intact mitochondrial membrane (%) Motility (%) ATP (nmol/109 sperm) ROS+ (%) Live sperm with intact mitochondrial membrane (%) ∗
P ≤ 0.05; ∗∗ P ≤ 0.01; NS—not significant.
NS NS –0.48∗∗ NS
Cryopreserved sperm characteristics 0.67∗∗∗ –0.79∗∗∗∗ 0.78∗∗∗ –0.57∗∗
–0.68∗∗∗∗ 0.82∗∗∗∗
P ≤ 0.001;
NS
–0.71∗∗∗∗
0.75∗∗∗∗ 0.69∗∗∗∗
∗∗∗
ATP (nmol/109 sperm)
0.73∗∗∗∗
–0.63∗∗
∗∗∗∗
P ≤ 0.0001.
results strongly suggest that MMP values can differentiate damage to mitochondria related to liquid storage and cryopreservation. In our study, turkey sperm viability did not change during liquid storage and was maintained at a high level (approximately 93%) for up to 48 h of storage. Those results are in agreement with previously published studies, where no changes in sperm viability or decline after 48 h of storage were reported for turkey semen (Donoghue and Donghue, 1997; Douard et al., 2000; Iaffaldano et al., 2005; Zaniboni and Cerolini, 2009; Rosato et al., 2012). No decline in sperm viability observed in our study strongly suggests that in the applied procedure of liquid storage, sperm maintain plasmalemma integrity, despite the decrease in motility and MMP. On the other hand, a dramatic decrease in sperm viability was observed after cryopreservation. After freezing/thawing, 30 to 41% of viable sperm was recorded for turkey sperm pellets (Blesbois et al., 2005; Iaffaldano et al., 2011; Lemoine et al., 2011). Summing up, contrary to liquid storage, cryopreservation has dramatic effect on the integrity of turkey spermatozoa, accompanied by a significant decrease in sperm motility, MMP, and ATP content. In our study, we observed that the percentage of live sperm with intact MMP was negatively correlated (r = –0.71∗∗ , r = –0.63∗∗ ) with ROS+ sperm during storage and cryopreservation, respectively. It is known that mitochondrial electron leaks have a major impact on mitochondrial coupling efficiency and the production of ROS (Turrens, 2003). Therefore, we suggest
that sperm mitochondria damage can be responsible for increased ROS+ production in turkey semen. Our observation was supported by the results of a study of cryopreserved chicken semen, indicating that ROS production can be the result of mitochondrial dysfunction in fowl semen (Partyka et al. 2011a; Nguyen et al., 2015). Therefore, it can be assumed that the decline in mitochondrial activity is associated with the increase in ROS generation during liquid storage and cryopreservation of turkey semen. It is well known that a high concentration of polyunsaturated fatty acids in bird spermatozoa makes membranes sensitive to lipid peroxidation in the presence of ROS (Fujihara and Howarth, 1978; Surai et al., 1998; Cerolini et al., 2006). In our study, for the first time, we measured the specific intracellular ROS level in turkey sperm during storage and cryopreservation. About 10% of ROS+ sperm were already detected in fresh turkey semen. We observed that oxidative sperm damage occurred after 24 h of liquid storage. It can be suggested that after 24 h of storage, the intracellular ROS levels exceed the available antioxidant capacity of the spermatozoa. A serious oxidative stress, 74% sperm ROS+ was observed after freezing/thawing and turkey spermatozoa seem to be more susceptible to oxidative stress caused by cryopreservation compared with other fowl [(44 and 8% for chicken and goose, respectively (Partyka et al., 2011a)]. Spermatozoa can be protected from lipid peroxidation by the antioxidant system present in seminal plasma (Slowi´ nska et al., 2011; Partyka et al., 2012b). It seems that the antioxidant
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Liquid stored sperm characteristics Live sperm ROS+(%) with intact mitochondrial membrane (%) 0.38∗ –0.54∗∗ 0.67∗∗ –0.62∗
Viability (%)
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SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online Figure S1. Representative dot plots displaying live and dead spermatozoa populations during liquid storage and cryopreservation. Figure S2. Representative dot plots displaying mitochondrial membrane potential of turkey semen during liquid storage and cryopreservation. The positive control involved incubating the sperm cells in the presence of 50 μmol/L carbonyl cyanide mchlorophenylhydrazone for 15 min at 37◦ C.
Figure S3. Representative histograms displaying ROS+ positive and ROS– negative spermatozoa population during liquid storage and cryopreservation. The positive control was performed by incubating the sperm cells in the presence of 3% H2 O2 , a superoxide anion generator, for 1 h at 37◦ C.
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system of turkey sperm is insufficient for protecting sperm during storage and cryopreservation. Perhaps, the dilution of semen, which is necessary for short-term storage and cryopreservation, leads to a decrease in the antioxidant protection of spermatozoa, resulting in higher ROS+ production in semen during storage, described previously for mammals frozen/thawed semen (Bilodeau et al., 2000; Cheema et al., 2009). In our study, we observed that equilibration affected only the ATP content of turkey spermatozoa. The only available data concerning effect of equilibration on semen quality refer to chicken. Similarly to our results, no effect of equilibration on motility and viability was observed (Roushdy et al., 2014; Kuzlu and Taskin, 2017). However, the negative effect of equilibration on sperm morphology was also reported (Chalah et al., 1999; Tselutin et al., 1999). Observed in our study, the decrease of ATP content without distribution of mitochondria function after equilibration is difficult to explain. Perhaps the rates of ATP hydrolysis already exceeded the rates of ATP production during this cryopreservation step. Further detailed studies concerning determination of all components of energy charge (ATP, adenosine diphosphate, and adenosine monophosphate) are necessary to test this hypothesis. Nevertheless, our results strongly suggest that the loss of ATP in cryopreserved spermatozoa has already commenced before freezing/thawing. The development of procedures aimed to counteract this ATP loss may lead to an improvement of cryopreservation procedures in turkey. In conclusion, a significant loss of the MMP and disturbance in sperm ATP production during semen storage can be the main reason for the decline in sperm motility. The disturbance of mitochondria activity during storage seems to be associated with the increase in oxidative stress in turkey semen. Therefore, MMP seems to be a useful marker of semen quality during storage. Turkey sperm mitochondria appear to be very sensitive to cryodamage. A decrease of energy production in turkey spermatozoa, visible as a low percentage of sperm with intact MMP and a low level of ATP after freezing/thawing, associated with high ROS generation, could be responsible for the low fertilizing ability of cryopreserved turkey semen.
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Nguyen, T. M., F. Seigneurin, P. Froment, Y. Combarnous, and E. Blesbois. 2015. The 5’-AMP-activated protein kinase (AMPK) is involved in the augmentation of antioxidant defenses in cryopreserved chicken sperm. PLoS One. 29:e0134420. Partyka, A., E. L ukaszewicz, and W. Ni˙za´ nski. 2011a. Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen. Theriogenology. 15:843–850. Partyka, A., E. L ukaszewicz, and W. Ni˙za´ nski. 2012a. Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen. Theriogenology. 77:1497–1504. Partyka, A., E. Lukaszewicz, and W. Ni˙za´ nski. 2012b. Lipid peroxidation and antioxidant enzymes activity in avian semen. Anim. Reprod. Sci. 134:184–190. Partyka, A., E. Lukaszewicz, W. Ni˙za´ nski, and J. Twardo´ n. 2011b. Detection of lipid peroxidation in frozen-thawed avian spermatozoa using C11-BODIPY581/591/591). Theriogenology. 75:1623– 1629. Partyka, A., W. Niza´ nski, and E. L ukaszewicz. 2010. Evaluation of fresh and frozen-thawed fowl semen by flow cytometry. Theriogenology. 74:1019–1027. Pel´ aez, J., and J. A. Long. 2007. Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy. J. Androl. 28:342–352. Rosato, M. P., G. Centoducati, M. P. Santacroce, and N. Iaffaldano. 2012. Effects of lycopene on in vitro quality and lipid peroxidation in refrigerated and cryopreserved turkey spermatozoa. Br. Poult. Sci. 53:545–552. Roushdy, K., M. A. El-Sherbieny, F. A. Abd El-Gany, and M. A. ELSayed. 2014. Semen cryopreservation for two local chicken strains as a tool for conservation of Egyptian local genetic resources. Egypt. Poult. Sci. 34:607–618. Sexton, T. J., and A. F. Giesen. 1983. Beltsville poultry semen extender. 8. Factors affecting turkey semen held six hours at 15 C. Poult. Sci. 62:1063–1068. Slowi´ nska, M., E. Liszewska, G. J. Dietrich, and A. Ciereszko. 2012. Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo). Theriogenology. 15:1065–1077. Slowi´ nska, M., G. J. Dietrich, E. Liszewska., K. Kozlowski, J. Jankowski, and A. Ciereszko. 2013. Effect of dialysis on the proacrosin/acrosin system and motility of turkey ( Meleagris gallopavo ) spermatozoa during liquid storage. Br. Poult. Sci. 54: 661–668. Slowi´ nska, M., J. Jankowski, G. J. Dietrich, H. Karol, E. Liszewska, J. Glogowski, K. Kozlowski, K. Sartowska, and A. Ciereszko. 2011. Effect of organic and inorganic forms of selenium in diets on turkey semen quality. Poult. Sci. 90:181–190. Surai, P. F., E. Blesbois, I. Grasseau, T. Chalah, J. P. Brillard, G. J. Wishart, S. Cerolini, and N. H. Sparks. 1998. Fatty acid composition, glutathione peroxidase and superoxide dismutase activity and total antioxidant activity of avian semen. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 120:527– 533. Thurston, R. 1995. Storage of poultry semen above freezing for twenty-four to forty-eight hours. Proc. The First International Symposium on Artificial Insemination of Poultry, Illinois, Savoy. Tselutin, K., F. Seigneurin, and E. Blesbois. 1999. Comparison of cryoprotectants and methods of cryopreservation of fowl spermatozoa. Poult. Sci. 78:586–590. Turrens, J. F. 2003. Mitochondrial formation of reactive oxygen species. J. Physiol. 552:335–344. Zaniboni, L., and S. Cerolini. 2009. Liquid storage of turkey semen: changes in quality parameters, lipid composition and susceptibility to induced in vitro peroxidation in control, n-3 fatty acids and alpha-tocopherol rich spermatozoa. Anim. Reprod. Sci. 112:51– 65.
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Donoghue, A. M., and G. J. Wishart. 2000. Storage of poultry semen. Anim. Reprod. Sci. 62:213–232. Douard, V., D. Hermier, and E. Blesbois. 2000. Changes in turkey semen lipids during liquid in vitro storage. Biol. Reprod. 63:1450– 1456. Douard, V., D. Hermier, M. Magistrini, C. Labb´e, and E. Blesbois. 2004. Impact of changes in composition of storage medium on lipid content and quality of turkey spermatozoa. Theriogenology. 61:1–13. Douard, W., D. Hermier, C. Labbe, M. Magistrini, and E. Blesbois. 2005. Role of seminal plasma in damage to turkey spermatozoa during in vitro storage. Theriogenology. 63:126–137. Folgerø, T., K. Bertheussen, S. Lindal, T. Torbergsen, and P. Oian. 1993. Andrology: mitochondrial disease and reduced sperm motility. Hum. Reprod. 8:1863–1868. Fujihara, N., and B. Howarth, Jr. 1978. Lipid peroxidation in fowl spermatozoa. Poult. Sci. 57:1766–1768. Iaffaldano, N., M. Di Iorio, S. Cerolini, and A. Manchisi. 2016. Overview of turkey semen storage: focus on cryopreservation – a review. Ann. Anim. Sci. 16:961–974. Iaffaldano, N., L. Romagnoli, A. Manchisi, and M. P. Rosato. 2011. Cryopreservation of turkey semen by the pellet method: effects of variables such as the extender, cryoprotectant concentration, cooling time and warming temperature on sperm quality determined through principal components analysis. Theriogenology. 15:794–801. Iaffaldano, N., M. P. Rosato, A. Manchisi, G. Centoducati, and A. Meluzzi. 2005. Comparison of different extenders on the quality characteristics of turkey semen during storage. Proc. 16th ASPA Congress, Torino. Jamieson, B. G. M. 2006. Avian spermatozoa: structure and phylogeny. Pages 349–398 in Reproductive Biology and Phylogeny of Birds. Part A: Phylogeny, Morphology, Hormones and Fertilization. B. G. Jamieson, ed. CRC Press, Taylor & Francis Group, Abingdon, England King, L. M., D. R. Holsberger, and A. M. Donoghue. 2000. Correlation of CASA velocity an linearity parameters with sperm mobility phenotype in turkeys. J. Androl. 21:65–71. Kotlowska, M., G. Dietrich, M. Wojtczak, H. Karol, and A. Ciereszko. 2007. Effects of liquid storage on amidase activity, DNA fragmentation and motility of turkey spermatozoa. Theriogenology. 15:276–286. Kuzlu, M., and A. Taskin. 2017. The effect of different extenders on the sperm motility and viability of frozen turkey semen. Indian J. Anim. Res. 51:235–241. Lemoine, M., S. Mignon-Grasteau, I. Grasseau, M. Magistrini, and E. Blesbois. 2011. Ability of chicken spermatozoa to undergo acrosome reaction after liquid storage or cryopreservation. Theriogenology. 1:122–130. Long, J. A. 2006. Avian semen cryopreservation: what are the biological challenges? Poult. Sci. 85:232–236. Long, J. A., and T. L. Conn. 2012. Use of phosphatidylcholine to improve the function of turkey semen stored at 4 C for 24 hours. Poult. Sci. 91:1990–1996. Long, J. A., and H. D. Guthrie. 2006. Validation of a rapid, largescale assay to quantify ATP concentration in spermatozoa. Theriogenology. 65:1620–1630. Long, J. A., and M. Kramer. 2003. Effect of vitamin E on lipid peroxidation and fertility after artificial insemination with liquidstored turkey semen. Poult. Sci. 82:1802–1807. Long, J. A., P. H. Purdy, K. Zuidberg, S. J. Hiemstra, S. G. Velleman, and H. Woelders. 2014. Cryopreservation of turkey semen: effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching. Cryobiology. 68:371–378. Marchetti, C., G. Obert, A. Deffosez, P. Formstecher, and P. Marchetti. 2002. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum. Reprod. 17:1257–1265.
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ABSTRACT The extensive use of artificial insemination in turkeys has led to the development of in vitro semen storage. However, fertility rates from liquid stored and frozen/thawed turkey semen are still unsatisfactory. The aim of the study was to assess spermatozoa viability, mitochondrial membrane potential (MMP), and reactive oxygen species production (ROS) in liquid stored and cryopreserved turkey semen with the use of flow cytometry. Moreover, motility and adenosine triphosphate (ATP) content in sperm were monitored at the same time to link flow cytometry data with sperm movement and energetics. Liquid storage led to a decrease in sperm motility (80.6 vs. 55.6%, for fresh and stored for 48 h), live sperm with an intact MMP (59.9 vs. 30.5% for fresh and stored for 48 h), and a 20-fold decrease in ATP content after 24 h of storage. A 3-fold increase in ROS+ sperm was observed after 48 h of storage (9.3 vs. 26.8% for fresh and stored for 48 h). Semen equili-
bration before cryopreservation affected only ATP content. However, freezing/thawing led to a dramatic decrease in all of the studied semen quality parameters. A 5-fold decrease in live sperm with intact MMP (59.8 vs. 11.9%) and a 7-fold increase in sperm ROS+ (10.8 vs. 74.4%) were recorded between fresh and frozen/thawed semen. The results strongly suggested that a significant loss of MMP and a disturbance in sperm ATP production during semen storage can be the main reason for the decline in sperm motility. The disturbance of mitochondria activity during storage seems to be associated with the increase in oxidative stress in turkey semen. Turkey sperm mitochondria also appear to be very sensitive to cryodamage. Diminished energy production in turkey spermatozoa, visible as the low percentage of sperm with an intact MMP and low level of ATP after freezing/thawing, which is associated with high ROS generation, could be responsible for the low fertilizing ability of cryopreserved turkey semen.
Key words: flow cytometry, semen, liquid storage, cryopreservation, turkey 2018 Poultry Science 97:3709–3717 http://dx.doi.org/10.3382/ps/pey209
INTRODUCTION The extensive use of artificial insemination in turkeys has led to the development of in vitro semen storage in a liquid form (Thurston, 1995; Blesbois and Brillard, 2007). The liquid storage of semen is of practical interest in the management of male turkeys. However, turkey semen cannot be stored for longer than 6 h without a loss of fertilizing ability, even if oxygenated and stored with appropriate diluents at a reduced temperature (Sexton and Giesen, 1983; Thurston, 1995). Turkey semen storage is accompanied by an irreversible reduction in motility, morphological integrity, and fertility C 2018 Poultry Science Association Inc. Received October 11, 2017. Accepted April 27, 2018. 1 This work was supported by the National Science Centre, Poland (2016/21/B/NZ9/03583) and funds appropriated to the Institute of Animal Reproduction and Food Research. 2 Corresponding author: Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research Polish Academy of Sciences Tuwima 10, 10-748 Olsztyn, Poland Tel.: +48 895393135; Fax: +48 895357421; E-mail: [email protected]
of spermatozoa (Clarke et al., 1982; Thurston, 1995; Donoghue and Donoghue, 1997). Many factors may contribute to the loss of fertilizing ability of spermatozoa stored in vitro, and factors affecting lipid biochemistry, including significant losses in the phospholipids of the plasma membrane, lipid peroxidation, and greater susceptibility to damage of cold-stored sperm membranes seem to be predominant (Donoghue et al., 1996; Douard et al., 2000, 2005; Breque et al., 2003; Long and Kramer, 2003). However, the mechanisms for sperm quality loss during liquid storage are currently unknown. Similarly to liquid storage, the poultry industry has not been able to take advantage of cryopreservation (Blesbois, 2007). The unique physiological features make avian spermatozoa highly sensitive to damage caused by cryopreservation (Long, 2006). Firstly, the elongated head with small cytoplasm content causes the less effective uptake of cryoprotectant by the cell (Donoghue and Wishart, 2000). Secondly, the avian sperm tail is approximately 8 times the length of the sperm head, which also predisposes poultry sperm to sensitivity to freezing damage (Donoghue and
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Institute of Animal Reproduction and Food Research, Polish Academy of Sciences in Olsztyn, Department of Gamete and Embryo Biology, Tuwima 10, 10-747 Olsztyn, Poland
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MATERIALS AND METHODS Birds, Housing, and Management Turkey BUT Big-6 line (British United Turkeys, Limited, Chester, England) was maintained under standard husbandry conditions at the Turkey Testing Farm in Frednowy, Poland. Males were photostimulated at 26 wk of age (14 L:10 D) and produced semen by 30 wk of age. Feed and water were available ad libitum. Individual semen samples designated for short-term storage and cryopreservation were collected by abdominal massage (Burrows and Quinns, 1937) during the routine collection of semen for artificial insemination.
Semen Storage Liquid storage Immediately after semen collection, individual semen samples were diluted (1:2) in Poultry Semen Extender (Ovodyl, IMV Technologies, I’Aigle, France). Samples of extended semen were stored for 48 h under refrigeration (4 to 7◦ C). Semen samples (n = 9) were shaken in aerobic conditions with an agitator (JW Electronic, Warsaw, Poland). Aliquots were removed from each sample 2.5, 24, and 48 h after semen collection for the measurements of semen quality parameters. At one time, no more than 3 semen samples were analyzed and every week 3 consecutive samples were collected and stored for analysis. Cryopreservation The cryopreservation procedure was performed according to the method developed by Iaffaldano et al. (2011) with some modification. Immediately after semen collection, individual semen samples were diluted (1:4) in Ovodyl. Samples of extended semen (n = 13) were cooled at 4◦ C for 60 min and then dimethylacetamide was added as a cryoprotectant to a final concentration of 8%. The diluted semen was gently inverted and equilibrated at 4◦ C for 5 min. Volumes of 80 μL of semen were plunged drop by drop directly into liquid nitrogen. The pellets were thawed by placing 1 pellet into a plastic 2.0 mL cryogenic tube and incubated in a water bath at 80◦ C for 60 s. Aliquots were removed from fresh semen, after the addition of dimethylacetamide (equilibration) and after freezing/thawing for the measurements of semen quality parameters. At one time, no more than 3 semen samples were collected for experiments and 3 consecutive samples were collected for further analysis each week.
Semen quality analysis Computer-Assisted Sperm Analysis Video recordings of motile sperm after 2.5, 24, and 48 h of liquid storage and in fresh, equilibrated and frozen/thawed semen were made using a microscope with a ×10 negative phase objective and a Sony CCD black and white video camera (SPT-M108CE). Video recordings were analyzed using Hobson Sperm Tracker (Hobson Vision Ltd, Baslow UK). MicroCell 2-chamber (20-μm depth of the chamber) slides (Conception Technologies, San Diego, CA) were used. The slides were mounted on a heated stage (41◦ C). Conditions of sperm dilutions, recordings, and Hobson Tracker settings followed those described by King et al. (2000) and Kotlowska et al. (2007) for turkey spermatozoa. Semen samples were incubated for 5 min in the motility buffer (50 mM Tris buffer, pH 7.4, 120 mM NaCl, 10 mM glucose, 2 mM CaCl2 ) at 39◦ C. Turkey semen was diluted 300 to 500 times to obtain 30 to 50 sperm on the screen at the start of motility. The motility buffer was supplemented with 0.5% BSA to prevent the adherence of spermatozoa to the glass slides. Flow Cytometry Flow cytometry analyses were performed using a Muse Cell Analyzer (EMD Millipore,
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Wishart, 2000; Long, 2006). Damage occurring during freeze-thawing first concerns the plasma membrane, mitochondria, and acrosome region and the nuclei (Blesbois, 2007). Such damage affects fertility and the duration of fertility, which is a very important factor in birds due to the long-term semen storage in the uterovaginal glands of the female (Blesbois and Brillard, 2007). Extending the knowledge concerning sperm cryoinjuries is a prerequisite for a better understanding of damage to sperm and the improvement of cryopreservation protocols for turkey semen. Recently, flow cytometry has gained popularity for avian semen quality analysis (Donoghue et al., 1995; Blanco et al., 2000; Christensen et al., 2004). Using flow cytometry, the large number of spermatozoa (>10 0000) is analyzed in a small volume of sample in a short time with the use of fluorescent probe allowing for observation of physical characteristics such as cell size, shape, and also any component or function of the spermatozoa. To date, the flow cytometry was applied for the comprehensive analysis of fresh and frozen-thawed fowl semen quality (Partyka et al., 2010; 2011a,b, 2012a). Plasma membrane integrity, acrosome integrity, mitochondrial function, and chromatin status, as well as lipid peroxidation, were assessed in fresh and cryopreserved semen of rooster and goose (Partyka et al., 2011a,b). However, only restricted analyses, regarding the measurements of sperm viability, membrane integrity, and the carbohydrate content of sperm glycocalyx, have been performed in turkey semen (Donoghue et al., 1995; Donoghue and Donoghue, 1997; Pel´ aez and Long, 2007; Long and Conn, 2012). Taking into account that fertility rates from liquid stored and frozen/thawed turkey semen have been consistently lower than that from stored chicken semen, there is a need for more detailed flow cytometry analyses of turkey semen. The aim of the present study was to assess spermatozoa viability, mitochondrial membrane potential (MMP), and reactive oxygen species production (ROS) in liquid stored and cryopreserved turkey semen with the use of flow cytometry. Moreover, motility and adenosine triphosphate (ATP) content in sperm were monitored at the same time to link flow cytometry data with sperm movement and energetics.
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performed, containing only sperm with a depolarized membrane. The positive control was performed by incubating the sperm cells in the presence of 50 μmol/L carbonyl cyanide m-chlorophenylhydrazone (SigmaAldrich, St. Louis, MO) for 15 min at 37◦ C according to Marchetti et al. (2002). ATP Concentration ATP concentration in turkey spermatozoa was measured using the ATP Colorimetric/Fluorometric Assay Kit (Sigma-Aldrich). Samples were prepared according to the method of Long and Guthrie (2006). Sample preparation included phosphatase inhibitor cocktail 2 (Sigma-Aldrich) treatment, ATP extraction (boiling water bath, 10 min), and ATP assay. ATP concentration was determined by phosphorylating glycerol, resulting in a fluorometric (λex = 535/λem = 587 nm) product proportional to the amount of ATP present.
Statistical Analysis The results are presented as mean ± SD. All analyses were performed at a significance level of P < 0.05 using GraphPad Prism software v.6.02 (GraphPad Software Inc. San Diego, CA). A 1-way ANOVA, followed by the Tukey test for post hoc comparison of means and Student’s t test, was used for analysis. For statistical procedures, the percentage data were normalized by arcsine square root transformation. Correlations between sperm quality parameters during liquid storage and cryopreservation were calculated using the Spearman correlation test.
RESULTS Motility and Motility Characteristics A steady decline in the percentage of sperm motility was observed during the storage; a significant decrease occurred after 48 h. No changes in other sperm motility characteristics related to both speed of movement (VCL, VSL and VAP) and trajectory of movement (BCF, ALH, LIN) were found at the same time (Table 1). Equilibration did not cause decrease in sperm motility and sperm motility characteristics (Table 1). A 4-fold decrease in the percentage of sperm motility was found in frozen/thawed semen. Among all sperm motility characteristics, only VSL was decreased after freezing/thawing (Table 1).
Viability No changes in sperm viability were found during 48 h of storage (Figure 1A, Supplementary Figure S1). The sperm viability was maintained at a high level (approximately 93%) for up to 48 h of storage. Equilibration did not affect sperm viability (Figure 1A’). The sperm viability in fresh and equilibrated
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Billerica, USA). Viability was assessed using Muse Count and Viability Reagent (EMD Millipore) according to the manufacturer’s protocol. A membranepermeant DNA-staining dye stained all cells with a nucleus, which is then used to discriminate cells with a nucleus from debris and non-nucleated cells. A DNAbinding Muse dye based on 7-aminoactinomycin D (7-AAD) stains cells that have lost their membrane integrity, allowing the dye to stain the nucleus of dead and dying cells. This parameter was displayed as viability and was used to discriminate viable (live cells that do not stain) from non-viable (dead or dying cells that stain) cells. Semen was diluted in PBS to obtain concentrations in the range of 1 × 105 to 1 × 107 sperm/mL. Subsequently, 20 μL of each semen suspension was mixed with 380 μL of Muse Count and Viability Reagent and incubated for 5 min at room temperature. The ROS production of spermatozoa was determined using a Muse Oxidative Stress Kit (EMD Millipore) according to the manufacturer’s protocol. Semen was diluted in 1X Assay Buffer to obtain concentrations in the range of 1 × 106 to 1 × 107 sperm/mL and then 10 μL of semen suspension was mixed with 190 μL of Muse Oxidative Stress working solution and incubated at 37◦ C for 30 min. The Muse Oxidative Stress Reagent is based on dihydroethidium (DHE), a well-characterized reagent that has been used extensively to detect ROS in cellular populations (Bindokas et al., 1996). Upon reaction with superoxide anions, DHE undergoes oxidation to form the DNA-binding fluorophore ethidium bromide or a structurally similar product which intercalates with DNA, resulting in red fluorescence. Two populations of cells were distinguished in the assay: ROS–, live cells, and ROS+, cells exhibiting ROS. To accurately determine the dividing point between ROS– and ROS+ populations, a positive control setup was performed, containing completely oxidized sperm cells. The positive control involved incubating sperm cells in the presence of 3% H2 O2 , a superoxide anion generator, for 1 h at 37◦ C. The MMP was determined using Muse MitoPotential Kit (EDM Millipore) according to the manufacturer’s protocol. Semen was diluted in 1X Assay Buffer to obtain a concentration in the range of 1 × 104 to 5 × 105 sperm/mL and then 100 μL of semen suspension was mixed with 95 μL of Muse MitoPotential working solution and incubated at 37◦ C for 20 min. Next, 5 μL of Muse 7-AAD was added to the sample and incubated at room temperature for 5 min. The Muse MitoPotential Reagent is based on MitoPotential Dye, a cationic, lipophilic dye, which detects changes in the MMP, and 7-AAD as an indicator of cell death. Four populations of cells were distinguished in the assay: live cells with depolarized MMP; live cells with intact MMP; dead cells with depolarized MMP; and dead cells with intact MMP. To accurately determine the dividing point between live cells with intact and depolarized mitochondrial membranes, a positive control setup was
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Table 1. Effect of time of liquid storage and cryopreservation on turkey spermatozoa motility and motility characteristics. Computer-assisted sperm analysis Liquid storage
Cryopreservation Motility (%) VCL (μ m/s) VSL (μ m/s) VAP (μ m/s) LIN (%) BCF (Hz) ALH (μ m)
80.6 153.5 84.5 120.4 53.3 10.7 8.0
Time of storage (h) 24 5.1a 71.6 ± 13.9a 55.6 10.2 150.5 ± 8.6 151.3 15.7 94.4 ± 12.9 98.0 15.6 122.1 ± 10.9 123.8 6.7 60.7 ± 6.9 63.1 1.3 11.5 ± 1.3 11.3 1.3 6.9 ± 1.8 6.4
Fresh 78.1 141.0 81.6 105.0 52.9 11.2 6.5
± ± ± ± ± ± ±
48 ± ± ± ± ± ± ±
7.3b 7.5 12.2 9.2 8.2 1.8 1.2
Equilibrated Frozen/thawed
10.6 74.8 ± 15.5a 19.6 ± 10.0b 16.4 143.8 ± 18.4 130.9 ± 13.2 21.5a,b 89.2 ± 24.2a 66.2 ± 26.0b 20.7 109.9 ± 22.7 95.4 ± 21.0 10.3 57.3 ± 12.0 47.1 ± 15.1 2.0 12.58 ± 2.7 10.33 ± 2.9 1.6 6.0 ± 1.2 6.9 ± 1.7 a
Data represent mean values ± SD a,b Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation. VSL = straight-line velocity; VCL = curvilinear velocity; VAP = average path velocity; LIN = linearity; BCF = beat-cross frequency; ALH = amplitude of lateral head displacement.
semen was maintained at a high level (about 90%). However, a 4-fold decrease in sperm viability was found after freezing/thawing (Figure 1A’, Supplementary Figure S1).
Mitochondrial Membrane Potential Two populations of live sperm were distinguished in fresh semen: live sperm with an intact MMP (approximately 60%) and live sperm with a depolarized MMP (approximately 30%). Storage significantly reduced the percentage of live sperm with an intact MMP at 24 and 48 h (Figure 2A). On the other hand, an increase in the percentage of live sperm with a depolarized MMP was observed after 24 and 48 h of storage (Figure 2B, Supplementary Figure S2). No changes in depolar-
Reactive Oxygen Species Fresh semen was characterized by the presence of sperm cells exhibiting ROS (approximately 10%). An increase in the percentage of sperm undergoing oxidative stress by 2- and 2.5-fold was found after 24 and 48 h of storage, respectively (Figure 3A, Supplementary Figure S3). Equilibration did not affect the percentage of ROS+ sperm. An almost 7-fold increase in the percentage of sperm undergoing oxidative stress was observed after freezing/thawing (Figure 3A’, Supplementary Figure S3).
ATP Content An almost 20-fold decrease in the ATP content was found after 24 h of storage (Figure 4A). A 2- and 40-fold decreases in ATP content were observed for equilibrated and frozen/thawed sperm, respectively (Figure 4A’).
Correlations between Sperm Quality Parameters Correlations between liquid stored sperm quality parameters are presented in Table 2. Motility, viability, life sperm with intact MMP, and ATP content were negatively correlated with ROS+. Live sperm with an intact
Figure 1. Effect of time of liquid storage (A) and cryopreservation (A’) on sperm viability. Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
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Motility (%) VCL (μ m/s) VSL (μ m/s) VAP (μ m/s) LIN (%) BCF (Hz) ALH (μ m)
2 ± ± ± ± ± ± ±
ized/dead sperm were observed (Figure 2C, Supplementary Figure S2). Equilibration did not affect the MMP (Figure 2A’and B’). A decreased percentage of both live sperm with intact MMP (5-fold, Figure 2A’) and sperm with depolarized MMP was observed after freezing/thawing (Figure 2B’). With the decrease of live sperm (with intact and depolarized mitochondrial membrane), a 9-fold increase of depolarized/dead sperm was observed (Figure 2C’, Supplementary Figure S2).
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MMP were positively correlated with motility and ATP content. Correlations between cryopreserved sperm quality parameters are presented in Table 2. Motility, viability, live sperm with an intact MMP, and ATP content were negatively correlated with ROS+. Viability, live sperm with an intact MMP, and ATP content were positively correlated with motility. Viability and live sperm with intact mitochondrial membrane were positively correlated with ATP content.
DISCUSSION In our study, a steady decline in the percentage of sperm motility was observed during storage; however, a significant decrease occurred after 48 h of storage. Those results are in agreement with previously published studies. The decline in semen motility is usually observed after 24 h (Douard et al., 2000, 2004; Slowi´ nska et al., 2012, 2013) or 48 h of storage with decrease to 40% of sperm motility (Douard et al., 2000, 2004; Iaffaldano et al., 2005; Kotlowska et al., 2007;
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Figure 2. Effect of time of liquid storage (A, B, C) and cryopreservation (A’, B’, C’) on live sperm with intact (A, A’), depolarized mitochondrial membrane (B, B’), and dead sperm with depolarized mitochondrial membrane (C, C’). Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
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Figure 4. Effect of time of liquid storage (A) and cryopreservation (A’) on sperm ATP concentration. Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
Zaniboni and Cerolini, 2009). Contrary to liquid storage, a dramatic effect on sperm motility was observed after cryopreservation. Approximately 20% of sperm motility was obtained after freezing/thawing in our study, which is in the range of 15 to 60% of sperm motility obtained after the cryopreservation of turkey semen (Iaffaldano et al., 2011, 2016; Lemoine et al., 2011; Slowi´ nska et al., 2012; Long et al. 2014). The application of computer-assisted sperm analysis for monitoring sperm motility allowed us to confirm that the procedure of liquid semen storage and cryopreservation produce a different range of decline in the percentage of sperm motility. The decline in sperm motility observed in our study during storage correlated (r = 0.38∗ ) with the decrease in MMP, measured for the first time for turkey semen. It is known that sperm motility is primarily dependent on mitochondrial function (Auger et al., 1989; Folgerø et al., 1993). Mitochondria, located in the sperm midpiece, generate a major part of the ATP required for sperm motility (Jamieson, 2006; Agnihotri et al., 2016). A significant loss of the MMP and a disturbance in sperm ATP production during semen storage, as observed in our study, can be the indicators for semen
quality. Therefore, it can be suggested that a disturbance to energy metabolism can be the reason of decline in sperm motility during turkey semen storage. It has to be mentioned that a decrease in mitochondrial potential after 24 h of storage appeared to be more pronounced (decrease by 33%) than a decrease in sperm motility (decrease by 11%). For this reason, MMP can be a potential marker of semen quality during storage. This suggestion is supported by the ATP decrease during liquid storage observed in our study and the studies of Douard et al. (2004) and Long and Guthrie (2006). It is known that poultry sperm mitochondria are sensitive to the freeze/thaw process and therefore ATP production is seriously compromised after cryopreservation (Long, 2006). In our study, we observed that turkey sperm mitochondria were also affected by cryopreservation. Firstly, we observed a 5-fold decrease in the population of live sperm with intact MMP and secondly the 40-fold decrease of ATP content of sperm after cryopreservation. A decrease in energy production in turkey spermatozoa, visible in a low percentage of sperm with an intact MMP and a low level of ATP after freezing/thawing, could be responsible for the low fertilizing ability of cryopreserved turkey semen. Our
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Figure 3. Effect of time of liquid storage (A) and cryopreservation (A’) on ROS+ sperm. Data represent mean values ± SD. Different letters indicate statistical significance at P ≤ 0.05 among storage time and cryopreservation.
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FLOW CYTOMETRY OF TURKEY SEMEN Table 2. Correlations between liquid stored and cryopreserved sperm characteristics of turkey semen.
Motility (%) ATP (nmol/109 sperm) ROS+ (%) Live sperm with intact mitochondrial membrane (%) Motility (%) ATP (nmol/109 sperm) ROS+ (%) Live sperm with intact mitochondrial membrane (%) ∗
P ≤ 0.05; ∗∗ P ≤ 0.01; NS—not significant.
NS NS –0.48∗∗ NS
Cryopreserved sperm characteristics 0.67∗∗∗ –0.79∗∗∗∗ 0.78∗∗∗ –0.57∗∗
–0.68∗∗∗∗ 0.82∗∗∗∗
P ≤ 0.001;
NS
–0.71∗∗∗∗
0.75∗∗∗∗ 0.69∗∗∗∗
∗∗∗
ATP (nmol/109 sperm)
0.73∗∗∗∗
–0.63∗∗
∗∗∗∗
P ≤ 0.0001.
results strongly suggest that MMP values can differentiate damage to mitochondria related to liquid storage and cryopreservation. In our study, turkey sperm viability did not change during liquid storage and was maintained at a high level (approximately 93%) for up to 48 h of storage. Those results are in agreement with previously published studies, where no changes in sperm viability or decline after 48 h of storage were reported for turkey semen (Donoghue and Donghue, 1997; Douard et al., 2000; Iaffaldano et al., 2005; Zaniboni and Cerolini, 2009; Rosato et al., 2012). No decline in sperm viability observed in our study strongly suggests that in the applied procedure of liquid storage, sperm maintain plasmalemma integrity, despite the decrease in motility and MMP. On the other hand, a dramatic decrease in sperm viability was observed after cryopreservation. After freezing/thawing, 30 to 41% of viable sperm was recorded for turkey sperm pellets (Blesbois et al., 2005; Iaffaldano et al., 2011; Lemoine et al., 2011). Summing up, contrary to liquid storage, cryopreservation has dramatic effect on the integrity of turkey spermatozoa, accompanied by a significant decrease in sperm motility, MMP, and ATP content. In our study, we observed that the percentage of live sperm with intact MMP was negatively correlated (r = –0.71∗∗ , r = –0.63∗∗ ) with ROS+ sperm during storage and cryopreservation, respectively. It is known that mitochondrial electron leaks have a major impact on mitochondrial coupling efficiency and the production of ROS (Turrens, 2003). Therefore, we suggest
that sperm mitochondria damage can be responsible for increased ROS+ production in turkey semen. Our observation was supported by the results of a study of cryopreserved chicken semen, indicating that ROS production can be the result of mitochondrial dysfunction in fowl semen (Partyka et al. 2011a; Nguyen et al., 2015). Therefore, it can be assumed that the decline in mitochondrial activity is associated with the increase in ROS generation during liquid storage and cryopreservation of turkey semen. It is well known that a high concentration of polyunsaturated fatty acids in bird spermatozoa makes membranes sensitive to lipid peroxidation in the presence of ROS (Fujihara and Howarth, 1978; Surai et al., 1998; Cerolini et al., 2006). In our study, for the first time, we measured the specific intracellular ROS level in turkey sperm during storage and cryopreservation. About 10% of ROS+ sperm were already detected in fresh turkey semen. We observed that oxidative sperm damage occurred after 24 h of liquid storage. It can be suggested that after 24 h of storage, the intracellular ROS levels exceed the available antioxidant capacity of the spermatozoa. A serious oxidative stress, 74% sperm ROS+ was observed after freezing/thawing and turkey spermatozoa seem to be more susceptible to oxidative stress caused by cryopreservation compared with other fowl [(44 and 8% for chicken and goose, respectively (Partyka et al., 2011a)]. Spermatozoa can be protected from lipid peroxidation by the antioxidant system present in seminal plasma (Slowi´ nska et al., 2011; Partyka et al., 2012b). It seems that the antioxidant
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Liquid stored sperm characteristics Live sperm ROS+(%) with intact mitochondrial membrane (%) 0.38∗ –0.54∗∗ 0.67∗∗ –0.62∗
Viability (%)
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SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online Figure S1. Representative dot plots displaying live and dead spermatozoa populations during liquid storage and cryopreservation. Figure S2. Representative dot plots displaying mitochondrial membrane potential of turkey semen during liquid storage and cryopreservation. The positive control involved incubating the sperm cells in the presence of 50 μmol/L carbonyl cyanide mchlorophenylhydrazone for 15 min at 37◦ C.
Figure S3. Representative histograms displaying ROS+ positive and ROS– negative spermatozoa population during liquid storage and cryopreservation. The positive control was performed by incubating the sperm cells in the presence of 3% H2 O2 , a superoxide anion generator, for 1 h at 37◦ C.
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system of turkey sperm is insufficient for protecting sperm during storage and cryopreservation. Perhaps, the dilution of semen, which is necessary for short-term storage and cryopreservation, leads to a decrease in the antioxidant protection of spermatozoa, resulting in higher ROS+ production in semen during storage, described previously for mammals frozen/thawed semen (Bilodeau et al., 2000; Cheema et al., 2009). In our study, we observed that equilibration affected only the ATP content of turkey spermatozoa. The only available data concerning effect of equilibration on semen quality refer to chicken. Similarly to our results, no effect of equilibration on motility and viability was observed (Roushdy et al., 2014; Kuzlu and Taskin, 2017). However, the negative effect of equilibration on sperm morphology was also reported (Chalah et al., 1999; Tselutin et al., 1999). Observed in our study, the decrease of ATP content without distribution of mitochondria function after equilibration is difficult to explain. Perhaps the rates of ATP hydrolysis already exceeded the rates of ATP production during this cryopreservation step. Further detailed studies concerning determination of all components of energy charge (ATP, adenosine diphosphate, and adenosine monophosphate) are necessary to test this hypothesis. Nevertheless, our results strongly suggest that the loss of ATP in cryopreserved spermatozoa has already commenced before freezing/thawing. The development of procedures aimed to counteract this ATP loss may lead to an improvement of cryopreservation procedures in turkey. In conclusion, a significant loss of the MMP and disturbance in sperm ATP production during semen storage can be the main reason for the decline in sperm motility. The disturbance of mitochondria activity during storage seems to be associated with the increase in oxidative stress in turkey semen. Therefore, MMP seems to be a useful marker of semen quality during storage. Turkey sperm mitochondria appear to be very sensitive to cryodamage. A decrease of energy production in turkey spermatozoa, visible as a low percentage of sperm with intact MMP and a low level of ATP after freezing/thawing, associated with high ROS generation, could be responsible for the low fertilizing ability of cryopreserved turkey semen.
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