Bielas et al. Acta Vet Scand (2017) 59:58 Acta Veterinaria Scandinavica DOI 10.1186/s13028-017-0325-9 RESEARCH Open Access Effect of long-term storage in Safe Cell extender on boar sperm DNA integrity + and other key sperm parameters Wiesław Bielas1, Wojciech Niżański1*, Agnieszka Partyka1, Anna Rząsa2 and Ryszard Mordak3 Abstract Background: There is some controversy about the extent of changes in different sperm cell features in stored boar semen, especially regarding the potential role of the DNA fragmentation assay for assessment of sperm fertiliz- ing ability. The aim of this study was to assess the effect of time of storage and the dynamic changes in sperm cell characteristics in normospermic boar semen stored in long-term extender, in order to determine the susceptibility to damage of particular structures of spermatozoa during cooling and storage at 17 °C for 240 h post collection. The study included five ejaculates from each of seven boars of the Polish Large White breed (n 35 ejaculates). The sperm = characteristics were assessed using a flow cytometer and a computer assisted sperm analyzer on samples at 0, 48, 96, 168 and 240 h post collection. Results: The sperm chromatin structure assay (SCSA) showed a significant abrupt increase (P < 0.01) in the DNA fragmentation index (%DFI) after 48 h of semen storage with only subtle changes thereafter, not exceeding 5% on average after 240 h of storage. The use of a combination of SYBR-14/PI stains did not reveal any significant changes in the percentage of live sperm cells up to 168 h of semen storage. A significant (P < 0.01) decrease in the percentage of live spermatozoa with intact acrosomes was observed after prolonged semen storage (168 h). A significant and progressive decrease in sperm motility was recorded during the whole period of semen storage. Conclusions: Storage of boar semen extended in long-term diluent at 17 °C for 48 h initially induced a decrease in the integrity of sperm DNA. This suggests that the structure of boar sperm DNA is susceptible to damage, especially during semen extension and at the beginning of sperm storage. These findings support the opinion that the SCSA test has only a low potential for routine assessment of boar semen preserved in the liquid state and for assessment of sperm quality changes during 10 days of semen preservation. Remarkably, the integrity of acrosomes and plasma membranes remained nearly unchanged for 7 days. Keywords: Boar, Spermatozoa, DNA fragmentation, Flow cytometry, Acridine orange, SCSA, CASA Background no breakthrough in the use of frozen boar semen [2], In the pig industry, the vast majority of sows are still sub- mainly due to the high sensitivity of boar spermatozoa to jected to artificial insemination (AI) with extended liquid cooling, freezing and thawing [3]. Commonly, boar sper- semen, so that preservation of the fertilizing capacity of matozoa are stored in liquid at 15–17 °C for routine use boar spermatozoa for several days remains an important in artificial insemination, but extenders for boar semen target for the industry [1]. Up until now, there has been for storage even at lower temperatures have also been available for a number of years [4–8]. Therefore, there is a growing interest in the development of new extend- *Correspondence: [email protected] ers and determination of optimal storage conditions for 1 Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life diluted boar spermatozoa. To preserve the quality of Sciences, pl. Grunwaldzki 49, 50-366 Wrocław, Poland spermatozoa in diluted boar semen during long-term Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bielas et al. Acta Vet Scand (2017) 59:58 Page 2 of 12 storage, the choice of long-term extender is critically A number of studies have indicated the potential of important [9]. Long-term extenders have certain advan- the SCSA for assessment of boar sperm quality [12, 26, tages: they allow for better organization in collection 31] and fertility [32–34]. The negative, damaging effect centers, support long-distance transport and provide the of semen handling and storage on boar sperm DNA has ability to conduct research on the semen before use [4]. previously been described, both with respect to liquid Unfortunately, even if extenders and lower temperatures storage [12, 16, 28, 34, 35] and frozen storage [36–38]. can prolong the lifespan of spermatozoa, physiologi- Dilution conditions [28, 35, 39], time of storage of liq- cal senescence of sperm cells still cannot be completely uid semen [12] and age of boars [40], as well as variation avoided. Aging-related changes may occur, consisting between ejaculates within boars [12, 32, 41], may be to of non-regulated, capacitation-like modifications [10], some extent implicated and responsible for the dam- structural and functional changes [1], oxidative processes age to boar sperm DNA integrity. Thus, damaged DNA in cell membranes [11] and damage to DNA integrity is considered to be one element responsible for reduced [12]. These changes can only be partially delayed by using capability of sperm cells to bind to the oviductal epithe- different extenders [13]. lium [41], as well as to underdevelopment of embryos Among the different indicators of sperm quality dur- [42, 43], and can lead to early embryonic or fetal death or ing storage, motility and integrity of the sperm plasma have a dramatic impact on health of the offspring [27]. In membrane have been the most evaluated characteristics recent studies, which investigated the relationship of flow in boars. Motility is assessed by means of computer- cytometric sperm integrity assessments with boar fertil- assisted semen analysis (CASA) [14], and sperm plasma ity, none of the individual membrane integrity variables membrane integrity through flow cytometry [1, 16]. was significantly related to fertility except the amount of These methods are good tools for sensitive assessment of DNA damage. These studies have shown that only sperm storage effects on sperm quality as well as for evaluation chromatin stability had a relationship with fertility from 7 of new extenders and preservation methods [16]. Both to 10 days and again from 14 to 15 days after ejaculation, sperm evaluation systems have been shown to be accu- dilution and long-term storage of semen [34]. Contradic- rate, precise and repeatable and have greatly improved tory results were obtained in some recent experiments the accuracy, objectivity and reproducibility of sperm showing that the level of DNA fragmentation in liquid evaluation [17, 18]. However, assessment of motility stored boar semen is very low for a long time [16, 19]. and sperm membrane integrity during storage only par- The aim of this study was to investigate changes in tially addresses the lowering of sperm fertilizing poten- the %DFI determined by SCSA along with changes in tial caused primarily by aging due to free radicals. Many key sperm parameters in boars to elucidate effects of factors including storage length, extender type, male boar and time of storage on sperm cell quality. During effect, boar age and breed affect boar sperm quality. With the study, semen was diluted and stored in Safe Cell , a + respect to this, assessment of the capacity of a specific long-term extender for 240 h at 17 °C. extender to maintain the quality of stored boar spermato- Methods zoa should also include DNA integrity [12, 19], acrosome intactness [13, 20], mitochondrial activity [21], bacterial Animals and semen collection contamination, pH determination [9], tyrosine phospho- Seven mature boars of the Polish Large White breed rylation [15] and apoptotic changes [18]. were used, ranging from 18 months to 3 years of age and One of the key features related to sperm fertility is selected according to the normal semen quality criteria, the integrity of the nuclear DNA, whose stability largely i.e., >50   108 total sperm cells per ejaculate, initial motil- × depends on the integrity of the chromatin. Therefore, ity >70%, and containing >70% morphologically normal some authors recommend assessment of chromatin spermatozoa. The boars in this study were routinely used integrity as a good, complementary and independent in our AI center as semen donors. Boars were randomly indicator of sperm quality [22]. Sperm DNA fragmenta- selected among all the AI males. Thirty-five ejaculates were tion tests, such as the DNA fragmentation index (%DFI), used in this experiment. Five ejaculates were collected from may provide a reliable guide to identify individuals that each male. Semen was collected once a week in one Pol- are at risk of failing to initiate a healthy pregnancy [23]. ish boar station, for 5 consecutive weeks. The sperm-rich There are several methods to assess sperm DNA frag- fraction of the ejaculate was collected using the gloved mentation, which have been used in the assessment of hand technique. Immediately after collection, the following boar spermatozoa. These methods are the TUNEL assay procedures were done: initial assessment of motility with a [24, 25], the sperm chromatin structure assay (SCSA) [26, phase-contrast microscope at 200 magnification; meas- × 27], the Comet assay [28] and the sperm chromatin dis- urement of sperm concentration with a SpermaCue pho- persion test (SCD) [29, 30]. tometer Porcine (Minitüb GmbH, Tiefenbach, Germany); Bielas et al. Acta Vet Scand (2017) 59:58 Page 3 of 12 and preparation of smears for subsequent staining with added. The working solution was obtained by diluting Giemsa stain [44] and routine sperm morphology assess- SYBR-14 in DMSO at a ratio of 1:49. Samples were mixed ment (1250 magnification). The sperm-rich fraction was and incubated at room temperature for 10 min and then × diluted with Safe Cell (IMV Technologies, l’Aigle, France) the cells were counterstained with 5 µL PI (2.4 mM work- + long-term extender to a final concentration of 30   106 ing solution) for 5 min before analysis [45–47]. × spermatozoa/mL to prepare conventional AI doses for fresh semen. The extended semen doses of 100 mL contain- Acrosome integrity ing approximately 3   109 sperm were packaged in plastic Acrosomal damage was assessed using PNA Alexa Fluor × bags. They were slowly cooled down to 17 °C and subse- 488 (Lectin from Arachis hypogaea, Merck Biosciences, quently transported to the Laboratory of Andrology at the Darmstadt, Germany). Ten microliter PNA Alexa Fluor Department of Reproduction, Faculty of Veterinary Medi- 488 working solution (1 µg/mL) was added to 500 µL of cine in Wrocław within 5 h after collection. In the labora- sperm sample (30   106 spermatozoa/mL) and incubated × tory, the semen doses were stored at 17 °C in a boar semen for 5 min at room temperature in the dark. Following incubator (Minitüb). Samples for computer assisted sperm incubation, the supernatant was removed by centrifuga- analysis and assessment in a flow cytometer were taken tion (500 g for 3 min) and the sperm pellets were re-sus- × immediately after arrival at the laboratory (0 h) and again pended in 500 µL of Safe Cell . Then, 5 µL of PI (2.4 mM + after 48, 96, 168 and 240 h of storage at 17 °C. working solution, AO; Life Technologies Ltd.) was added to samples before cytometric analysis [48]. Assessment of sperm cell characteristics Motility Assessment of chromatin status Sperm motion characteristics in extended semen were Sperm samples were diluted in Safe Cell diluent to a + evaluated using CASA (Hamilton-Thorne Sperm Ana- final concentration of 1    106 spermatozoa/mL. The × lyser IVOS version 12.2l, Hamilton Thorne Biosciences, suspension (200 µL) was subjected to brief acid dena- MA, USA), under 1.89   10 magnification. A 3 µL ali- turation by mixing with 400 µL of lysis solution [Triton × quot of semen was placed in a Leja4 analysis chamber X-100 0.1% (v/v), NaCl 0.15 M, HCl 0.08 M, pH 1.4], held (Leja, Nieuw-Vannep, Netherlands) at 35 °C and evalu- for 30 s and mixed with 1.2 mL acridine orange solution ated. Settings of the IVOS were the following: frame (AO; Life Technologies Ltd.) (6 µg AO/mL in a buffer: acquired 45, frame rate 60 Hz, minimum cell contrast citric acid 0.1 M, Na HPO 0.2 M, EDTA 1 mM, NaCl 2 4 46, minimum cell size 7, straightness threshold 45%, path 0.15 M, pH 6). After 3 min samples were aspirated into a velocity threshold 45 µ/s, path velocity cut off 20 µ/s, flow cytometer [49]. straight line velocity cutoff 5 µ/s, head size non-motile 7, head intensity non-motile 50, static head size 0.65–4.90, Assessment of mitochondrial activity static head intensity 0.50–2.50, static elongation 0–87. The percentage of spermatozoa with functional mito- Six fields randomly selected by a computer were analyzed chondria was estimated by combining fluorescent stains: for each semen sample. The motility parameters obtained Rhodamine 123 (R123; Life Technologies Ltd.) and PI. by the IVOS analyzer were: VAP (average path velocity, R123 solution (10 µL) was added to 500 µL of diluted µm/s), VSL (straight line velocity, µm/s), VCL (curvilin- sperm samples (50   106 spermatozoa/mL) and incu- × ear line velocity, µm/s), ALH (amplitude of lateral head bated for 20 min at room temperature in the dark. Sam- displacement, µm), BCF (beat cross frequency, Hz), LIN ples were then centrifuged at 500 g for 3 min and the × (linearity, %), MOT (total motility, %), PMOT (progres- sperm pellets were resuspended in 500 µL Safe Cell + sive motility, %), subpopulation of RAPID cells (veloc- extender. Then PI (2.4 mM working solution) was added ity > mean velocity of sperm population, %). CASA was as previously described [48]. set for analysis (5 microscopic views), more than 200 spermatozoa per sample were examined. Flow cytometry (FC) Measurements were performed on a FACSCalibur (Bec- Sperm membrane integrity ton–Dickinson, San Jose, CA, USA) flow cytometer. Sperm membrane integrity was assessed using dual flu- The fluorescent probes were excited by an Argon ion orescent probes, SYBR-14 and propidium iodide (PI) 488 nm laser. SYBR-14 fluorescence (cells with intact (Live/Dead Sperm Viability Kit, Life Technologies Ltd., plasma membranes), PNA Alexa Fluor 488 signal (cells Carlsbad, CA, USA). Samples with a concentration of with damaged acrosomes), and Rhodamine 123 fluores- 30   106 spermatozoa/mL were taken for the analysis. cence (cells with active mitochondria) were detected on × Portions (300 µL) of the samples were pipetted into cyto- detector FL2. PI fluorescence (cells with damaged plasma metric tubes and 5 µL of SYBR-14 working solution was membranes) was detected on detector FL1. Green Bielas et al. Acta Vet Scand (2017) 59:58 Page 4 of 12 fluorescence of acridine orange (double-stranded DNA) motile spermatozoa assessed subjectively in fresh semen was detected on the FL1 detector and red fluorescence of and the concentration of spermatozoa per mL were AO (single-stranded DNA) with detector FL3. This is the 76.6   6.1 and 516.5   106   134.2   106, respectively. ± × ± × standard protocol for flow cytometer analysis. The percentages of sperm cells with primary and second- Gates were set according to forward and side scatters ary defects of morphology were 12.9   4.6 and 3.1   1.7, ± ± to eliminate particles smaller than sperm in cell aggre- respectively (mean   SD). ± gates. For SYBR-14/PI, PNA Alexa Fluor 488/PI and Rhodamine 123/PI fluorochrome quadrants were set CASA and flow cytometric assessment of spermatozoa on dot plots of the logs of green fluorescent events (live Motility spermatozoa, damaged acrosomes, active mitochondria), A gradual decrease of MOT and PMOT of sperma- and red fluorescent events (dead spermatozoa) and dual tozoa was observed in samples stored in long-time staining [45, 47]. extender (Table 1). The decrease of MOT was signifi- The extent of DNA denaturation, expressed as the cant (P < 0.01) in all analysis periods, beginning from DNA fragmentation index (%DFI), was calculated based 48 h. The values for MOT of spermatozoa stored at 168 on the ratio of red/total (red   green) fluorescence for and 240 h were relatively low. Similarly, initial values + each sperm cell in the sample [22]. For each sample, two of PMOT were below 25%. There were no significant terms of DFI were evaluated: the percentage of sper- differences between PMOT at 0, 48 and 96 h. A sig- matozoa outside the main population with denatured nificant drop of PMOT at 168 h was noted (P < 0.001). DNA (%DFI) and the percentage of spermatozoa with an The initial mean value of STR was 40.9%   4.7 and LIN ± abnormally high DNA stainability (%HDS). The percent- was 20.4%   2.7 (mean   SD). STR and LIN increased ± ± age of HDS cells was calculated by setting the appropri- concomitantly with the decrease of ALH and PMOT. ate gate above the upper border of the main cluster of the A gradual decrease in the velocity of spermatozoa was sperm population with no detectable DNA denaturation, observed. A dramatic drop of VAP and the subpopula- mainly immature cells. tion of RAPID cells at 96 and 168 h of semen storage was Acquisitions were performed using the CellQuest 3.3 observed. A significant effect on all sperm motion char- software (Becton–Dickinson, San Jose, CA, USA). The acteristics was shown for time of storage (P < 0.0001), non-sperm events were gated out based on scatter prop- and with the exception of MOT and BCF, for the fac- erties and excluded from analysis. A total of 40   103 tors boar and interaction of boar and time (P < 0.05, × events (spermatozoa) were analyzed for each sample. P < 0.0001) (Table 1). Statistical analysis Viability The results obtained, presented as mean   SD of meas- The percentage of spermatozoa with an intact plasma ± urements on samples from 35 replicate determinations, membrane (i.e., live spermatozoa) was relatively high and were analyzed by ANOVA considering the time of stor- nearly unchanged up to 168 h of sperm storage (Table 2). age and boars as the main variables. When ANOVA At 240 h of storage a significant (P < 0.01) decrease in revealed a significant effect, values were compared by live cells was observed. The percentage of dead cells the least significant difference pairwise multiple com- increased (P < 0.01) earlier, i.e., at 168 h of sperm stor- parison post hoc test (Tukey’s test). Differences were age. The percentage of cells that exhibited a partly green considered to be significant if the calculated probability fluorescence and a partly red fluorescence (moribund, of their occurring by chance was <5% (P < 0.05). The sta- dying cells) remained unchanged during the whole stor- tistical model included the effect of time of storage and age period. the interaction between boar and time of storage. All percentage data were arc sin transformed to normalize Acrosome integrity unequal variances. The percentage of live cells with an intact acrosome The Spearman’s rank correlation coefficients were cal- began to decrease significantly (P < 0.01) at 168 h of stor- culated to measure the statistical dependence among all age (Table 2). However, even on the 10th day of storage variables, i.e., among all parameters assessed in the study almost 80% of sperm cells possessed intact acrosomes. at 0, 48, 96, 168 and 240 h of sperm storage. The percentage of live spermatozoa with damaged acrosomes remained constant during storage up to Results 240 h; a significant difference was detected only for the Characteristics of fresh semen factor boar (P < 0.0001). The percentage of dead sper- The mean volume of ejaculates collected from the boars matozoa with intact acrosomes remained unchanged was 264.2   47.4 mL. The percentage of progressively for 168 h of sperm storage. The value of this parameter ± Bielas et al. Acta Vet Scand (2017) 59:58 Page 5 of 12 Table 1 Motion characteristics of boar spermatozoa assessed by computer assisted semen analyzer (CASA) in semen stored for 240 h at 17 °C (mean   SD, n   35) ± = Spermatozoa Hours of incubation Source of variability 0 h 48 h 96 h 168 h 240 h Boar Time Interaction MOT (%) 77.3 12.4A 67.3 19.6B 55.7 19.0C 32.0 18.1D 20.8 15.3E 0.0083 <0.0001 0.2397 ± ± ± ± ± PMOT (%) 24.0 9.6A 21.2 10.1A 20.2 11.9A 11.3 7.4Ba 6.6 5.4Bb 0.0001 <0.0001 0.0040 ± ± ± ± ± VAP (µm/s) 99.0 17.9A 98.8 17.2A 79.1 17.1B 65.1 15.9C 53.2 20.4D 0.0004 0.0000 0.0390 ± ± ± ± ± VSL (µm/s) 38.2 6.4A 38.3 7.4A 35.8 8.4AB 32.5 5.9B 27.7 9.1C 0.0004 0.0000 0.0004 ± ± ± ± ± VCL (µm/s) 209.2 37.4A 209.8 35.7A 173.5 37.9B 143.3 34.3C 119.1 47.2D 0.0008 0.0000 0.0399 ± ± ± ± ± ALH (µm) 9.4 1.1A 9.6 0.9Aa 9.5 0.9A 8.9 1.3Ab 7.7 2.6B <0.0001 <0.0001 0.0001 ± ± ± ± ± BCF (Hz) 34.8 2.8A 34.4 3.1A 32.8 2.9A 33.1 3.2A 30.4 9.1B 0.1660 <0.0001 <0.0001 ± ± ± ± ± STR (%) 40.9 4.7A 41.0 4.6A 47.1 7.0Ba 52.2 8.4Bb 51.1 17.4B 0.0001 <0.0001 <0.0001 ± ± ± ± ± LIN (%) 20.4 2.7Aa 20.4 2.4Aa 22.9 3.4AC 25.6 4.5B 24.6 8.5BC <0.0001 <0.0001 <0.0001 ± ± ± ± ± RAPID (%) 63.5 16.4A 55.7 21.8A 41.7 19.6B 21.2 15.2Ca 12.5 10.6Cb 0.0039 <0.0001 0.0477 ± ± ± ± ± Characteristics assessed by CASA: MOT-percentage of motile spermatozoa; PMOT-percentage of progressively motile spermatozoa; VAP-average path velocity; VSL-straight line velocity; VCL-curvilinear velocity; ALH-amplitude of lateral head displacement; BCF-beat cross frequency; STR-straightness; LIN-linearity; RAPID- subpopulation of rapid cells Different superscripts within a row indicate significant differences a,b P < 0.05; A,B,C,D,E P < 0.01 Table 2 Plasma membrane integrity, acrosome integrity, sperm chromatin structure assay and mitochondrial activity of boar spermatozoa in semen stored for 240 h at 17 °C (mean   SD, n   35) ± = Spermatozoa (%) Hours of incubation Source of variability 0 h 48 h 96 h 168 h 240 h Boar Time Interaction Live (SYBR PI ) 88.8 8.2A 88.5 6.6A 88.1 7.7A 87.4 7.9A 85.5 7.7B <0.0001 0.0433 0.9981 + − ± ± ± ± ± Live with intact acrosome (PNA PI ) 82.5 7.5AB 85.0 6.1A 81.5 9.1AB 80.1 8.9B 78.6 8.0B <0.0001 0.0005 0.9987 − − ± ± ± ± ± %HDS 0.82 0.5 0.73 0.3 0.65 0.3 0.79 0.3 0.84 0.3 <0.0001 0.1407 0.8690 ± ± ± ± ± %DFI 3.55 2.7A 3.95 2.5B 4.14 2.4BC 4.40 2.4Ca 4.71 2.2Cb <0.0001 0.0200 0.9996 ± ± ± ± ± Live with active mitochondria (R PI ) 83.0 6.4 83.2 6.9 82.2 6.7 81.7 7.2 79.1 6.9 <0.0001 0.1188 0.9999 + − ± ± ± ± ± SYBR, SYBR-14; PI, propidium iodide; PNA, PNA Alexa Fluor 488 (lectin from Arachis hypogaea); PI, propidium iodide; %DFI (DNA fragmentation index)—the percentage of spermatozoa with DNA fragmentation; %HDS (high DNA stainability)—the percentage of spermatozoa with immature chromatin (less chromatin condensation); R, Rhodamine 123; PI, propidium iodide Different superscripts within a row indicate significant differences a,b P < 0.05; A,B,C P < 0.01 increased significantly (P < 0.05) at 240 h of sperm stor- abnormally high DNA stainability (%HDS), i.e., immature age whereas the percentage of sperm cells with damaged cells was similar at all times of sample analysis. plasma membranes and damaged acrosomes remained nearly constant during the whole period of sperm Mitochondrial activity storage. A gradual increase in the percentage of live spermato- zoa with inactive mitochondria was observed. There was Chromatin structure a significant difference (P < 0.05) between 0, 168 and The %DFI, describing the percentage of spermatozoa out- 240 h of storage (Table 2). No significant differences were side the main population with denatured DNA, increased noted in the percentage of live spermatozoa with active significantly (P < 0.01) within a short time after semen mitochondria during storage (P > 0.05) between 0 and collection and dilution, and was already apparent at 48 h 240 h. There were no significant differences between the of sperm storage (Table 2). At each time point of sperm percentages of dead sperm cells with active and inactive assessment, a significant increase in %DFI was observed. mitochondria in consecutive measurements. However, a A significant effect on sperm chromatin integrity was significant effect on these subpopulations of dead sperm shown for boar and time (P < 0.0001 and P < 0.05, cells was shown for boar and time of storage (P < 0.0001 respectively) The percentage of spermatozoa with an and P < 0.05, respectively). Bielas et al. Acta Vet Scand (2017) 59:58 Page 6 of 12 Relationships among boar sperm cell characteristics in extender at 17 °C. While extenders for boar semen for during storage storage at lower temperatures have been available for a In Fig. 1, the relationship between the %DFI and motil- number of years [4–8], these are not sufficiently effective ity (MOT and PMOT) and the viability, acrosome and for practical use in pig AI and further work is needed to plasma membrane integrity characteristics, is shown for produce efficient low temperature extenders. 35 ejaculates, representing different patterns of changes The decrease of MOT and PMOT is similar to values in these boar sperm cell parameters during storage. obtained by others [18, 50]. The percentages of motile Statistically significant correlations between motility sperm stored for 5 days in long-term extenders X-cell, parameters at consecutive hours of semen incubation Androstar and Mulberry III were 55.6, 49.9 and 80.5, (Figs. 2, 3) were observed. The experiment also revealed respectively [18]. many statistically significant correlations among the We found that the percentages of boar spermatozoa majority of the structural parameters of boar sperm cells with intact plasma membranes were usually more that during incubation. Moreover, the %DFI and %HDS were 80% and did not change significantly during storage for well correlated with structural parameters at 0 h of incu- 168 h. Similar results concerning sperm plasma mem- bation. After that only %DFI was strongly correlated with brane integrity in stored boar spermatozoa were obtained sperm structural features while %HDS lost that feature. by others [18]. At 240 h of incubation, neither parameter presented sig- The study revealed a relative stability of sperm acroso- nificant correlations with structural characteristics of mal membranes during storage of boar semen in the liq- sperm cells. Overall, there were no strong correlations uid state. It should be noted that there was a discrepancy between motility parameters and structural parameters between apparent loss of sperm motility and unchanged of boar spermatozoa stored in the liquid state. percentages of acrosome intact spermatozoa at consecu- tive measurements up to 168 h of semen storage. Discussion Based on a total of 35 ejaculates collected from seven Artificial insemination in pigs is mostly done using boar boars over a period of 5 weeks, 17.1% of the sperm semen preserved in the liquid state at 16–17 °C. There- samples showed >3%DFI, 11.4% showed >5%DFI, and fore, semen in the present study was diluted and stored 5.7% showed >10% of %DFI. Thus, our findings confirm 100 90 80 70 MOT 60 % PMOT 50 PSA- PI- 40 SYBR-14+/PI- 30 %DFI 20 10 0 0 h 48 h 96 h 168 h 240 h Fig. 1 Changes in motility, progressive motility, acrosome integrity, plasma membrane integrity and DNA fragmentation in spermatozoa stored for 240 h at 17 °C (mean SD, n 35). Characteristics assessed by computer assisted sperm analyzer: MOT-percentage of motile spermatozoa; PMOT- ± = percentage of progressively motile spermatozoa. Characteristics assessed by flow cytometer: PNA PI live spermatozoa with intact acrosome; − − SYBR-14 /PI live spermatozoa; %DFI the percentage of spermatozoa with DNA fragmentation + − Bielas et al. Acta Vet Scand (2017) 59:58 Page 7 of 12 0h VAP VSL VCL ALH BCF STR LIN MOT PROG RAPID Dead Morib Live PI+PNA- PI+PNA+ PI- PI- HDS DFI Dead Dead Live PNA- PNA+ inact act inact VAP VSL 0.70 VCL 0.97 0.69 ALH 0.58 0.34 0.70 BCF 0.39 0.14 0.30 -0.06 STR -0.51 0.10 -0.54 -0.53 -0.46 LIN -0.46 0.11 -0.53 -0.65 -0.36 0.95 MOT 0.46 0.58 0.34 -0.10 0.18 0.19 0.31 PROG 0.28 0.69 0.20 -0.18 -0.13 0.53 0.59 0.75 RAPID 0.68 0.81 0.60 0.17 0.19 0.04 0.13 0.85 0.80 Dead -0.22 -0.28 -0.17 -0.01 -0.24 0.14 0.10 -0.16 -0.11 -0.24 Morib -0.05 -0.13 -0.01 0.11 -0.03 -0.10 -0.12 -0.13 -0.15 -0.11 0.74 Live 0.10 0.13 0.08 0.02 0.32 -0.29 -0.22 0.01 -0.14 0.05 -0.76 -0.55 PI+PNA- 0.00 0.06 0.04 0.07 -0.11 0.06 0.07 -0.12 0.02 -0.03 0.56 0.52 -0.47 PI+PNA+ -0.36 -0.25 -0.27 0.08 -0.20 0.09 0.04 -0.37 -0.25 -0.42 0.23 0.14 0.07 0.21 PI-PNA- 0.05 -0.06 0.01 -0.04 0.05 -0.21 -0.16 0.00 -0.15 -0.03 -0.44 -0.48 0.45 -0.73 -0.40 PI-PNA+ 0.10 0.12 0.06 -0.13 0.24 0.11 0.10 0.31 0.28 0.27 -0.17 -0.03 -0.04 -0.36 -0.10 -0.25 HDS -0.36 -0.38 -0.28 0.02 -0.43 0.11 -0.01 -0.59 -0.28 -0.49 0.47 0.30 -0.51 0.19 0.21 -0.02 -0.32 DFI -0.13 -0.18 -0.06 0.15 -0.30 0.12 0.04 -0.22 -0.05 -0.15 0.73 0.77 -0.70 0.50 0.22 -0.52 -0.05 0.57 Dead -0.09 -0.22 0.01 0.13 -0.16 -0.13 -0.15 -0.24 -0.12 -0.19 0.53 0.37 -0.22 0.42 0.40 -0.43 -0.10 0.13 0.45 inact Dead act -0.29 -0.15 -0.29 -0.03 -0.17 0.25 0.21 -0.08 -0.03 -0.12 0.45 0.54 -0.56 0.20 -0.10 -0.12 -0.07 0.47 0.55 -0.05 Live inact -0.10 -0.08 -0.07 -0.01 -0.12 0.11 0.09 -0.04 0.02 -0.02 0.44 0.55 -0.55 0.35 -0.09 -0.31 -0.07 0.34 0.56 0.20 0.63 Live act 0.20 0.20 0.12 -0.07 0.28 -0.10 -0.06 0.15 0.04 0.12 -0.64 -0.59 0.58 -0.49 -0.16 0.47 0.07 -0.34 -0.68 -0.65 -0.52 -0.82 48h VAP VSL VCL ALH BCF STR LIN MOT PROG RAPID Dead Morib Live PI+PNA- PI+PNA+ PI- PI- HDS DFI Dead Dead Live PNA- PNA+ inact act inact VAP VSL 0.74 VCL 0.97 0.71 ALH 0.66 0.28 0.75 BCF 0.47 0.32 0.51 0.20 STR -0.48 0.17 -0.48 -0.54 -0.25 LIN -0.43 0.18 -0.48 -0.59 -0.41 0.92 MOT 0.67 0.71 0.57 0.04 0.39 -0.10 -0.03 PROG 0.45 0.86 0.39 -0.10 0.16 0.41 0.42 0.76 RAPID 0.76 0.83 0.67 0.13 0.40 -0.10 -0.04 0.94 0.81 Dead -0.22 -0.43 -0.17 0.13 -0.38 -0.28 -0.22 -0.39 -0.50 -0.38 Morib -0.20 -0.33 -0.20 -0.08 -0.24 -0.17 -0.13 -0.27 -0.33 -0.27 0.59 Live 0.05 0.15 0.05 -0.08 0.35 0.15 0.08 0.17 0.21 0.09 -0.76 -0.61 pi+pna- -0.05 0.00 -0.08 -0.09 -0.21 0.08 0.09 0.04 0.02 0.01 0.50 0.20 -0.48 pi+pna+ -0.48 -0.59 -0.45 -0.22 -0.29 0.02 0.01 -0.57 -0.52 -0.55 0.35 0.26 -0.10 -0.02 pi-pna- 0.26 0.27 0.23 0.02 0.35 -0.16 -0.17 0.37 0.25 0.35 -0.46 -0.33 0.47 -0.45 -0.55 pi-pna+ -0.11 -0.27 -0.06 0.15 -0.10 -0.02 -0.04 -0.28 -0.30 -0.26 0.09 0.13 -0.30 -0.10 0.34 -0.64 hds -0.26 -0.31 -0.28 -0.18 -0.46 -0.04 0.04 -0.31 -0.26 -0.26 0.26 0.38 -0.35 -0.09 0.51 -0.32 0.27 dfi -0.06 -0.15 -0.06 0.07 -0.40 -0.12 -0.08 -0.28 -0.28 -0.23 0.52 0.69 -0.56 0.30 0.24 -0.32 0.09 0.56 Dead -0.15 -0.24 -0.09 0.13 -0.10 0.00 -0.07 -0.35 -0.28 -0.33 0.59 0.49 -0.37 0.52 0.40 -0.57 0.16 -0.04 0.36 inact Dead act -0.08 -0.09 -0.11 -0.17 -0.31 -0.08 0.07 0.09 -0.06 0.08 0.32 0.47 -0.52 -0.05 -0.02 0.00 0.19 0.37 0.44 -0.09 Live inact 0.10 0.05 0.12 0.08 -0.08 -0.05 -0.05 0.05 0.03 0.10 -0.04 0.34 -0.15 -0.30 0.24 -0.12 0.30 0.61 0.33 -0.10 0.41 Live act 0.08 0.17 0.07 -0.02 0.25 0.00 -0.02 0.22 0.19 0.17 -0.45 -0.70 0.48 -0.18 -0.44 0.54 -0.36 -0.45 -0.65 -0.62 -0.41 -0.60 96h VAP VSL VCL ALH BCF STR LIN MOT PROG RAPID Dead Morib Live PI+PNA- PI+PNA+ PI- PI- HDS DFI Dead Dead Live PNA- PNA+ inact act inact VAP VSL 0.79 VCL 0.97 0.81 ALH 0.46 0.31 0.47 BCF 0.19 0.26 0.34 0.06 STR -0.27 0.27 -0.20 -0.34 0.09 LIN -0.28 0.21 -0.25 -0.47 -0.09 0.91 MOT 0.62 0.58 0.54 0.42 0.07 0.00 0.06 PROG 0.60 0.85 0.59 0.22 0.22 0.40 0.39 0.78 RAPID 0.80 0.77 0.74 0.47 0.11 -0.04 0.00 0.91 0.86 Fig. 2 Spearman’s correlation coefficients between all analyzed sperm parameters in boar spermatozoa stored for 240 h at 17 °C (n 35). Values in = bold font—statistically significant correlation (P < 0.05). VAP-average path velocity; VSL-straight line velocity; VCL-curvilinear velocity; ALH-amplitude of lateral head displacement; BCF-beat cross frequency; STR-straightness; LIN-linearity; mot-percentage of motile spermatozoa; PMOT-percentage of progressively motile spermatozoa; RAPID-subpopulation of rapid cells; Live—SYBR PI; PI PNA live with intact acrosome; HDS—%HDS (high + − − DNA stainability) the percentage of spermatozoa with immature chromatin; DFI—%DFI (DNA fragmentation index) percentage of spermatozoa with DNA fragmentation; Live act—live with active mitochondria (Rhodamine PI ) + − Bielas et al. Acta Vet Scand (2017) 59:58 Page 8 of 12 Dead 0.17 0.19 0.19 -0.14 0.12 -0.01 0.07 -0.03 0.01 0.08 Morib 0.06 0.15 0.13 -0.05 0.25 0.10 0.08 -0.10 0.03 -0.01 0.79 Live -0.07 -0.20 -0.11 0.13 -0.19 -0.10 -0.14 -0.13 -0.13 -0.16 -0.80 -0.74 PI+PNA- 0.21 0.24 0.24 0.07 0.25 0.08 0.06 0.13 0.17 0.18 0.64 0.49 -0.64 PI+PNA+ -0.04 0.18 -0.05 -0.55 -0.06 0.42 0.49 -0.11 0.22 -0.09 0.29 0.20 -0.06 0.06 PI-PNA- -0.11 -0.13 -0.13 0.19 -0.26 -0.18 -0.20 -0.18 -0.14 -0.15 -0.64 -0.66 0.74 -0.52 -0.33 PI-PNA+ 0.08 -0.11 0.09 -0.09 0.12 -0.11 -0.12 0.13 -0.10 0.09 0.13 0.23 -0.30 -0.17 -0.02 -0.61 HDS -0.20 -0.03 -0.17 -0.30 0.17 0.29 0.25 -0.12 0.03 -0.17 0.07 -0.12 0.08 -0.07 0.44 -0.03 -0.03 DFI -0.02 0.08 -0.05 -0.34 0.13 0.27 0.29 -0.09 0.06 -0.03 0.57 0.58 -0.59 0.41 0.35 -0.59 0.24 0.17 Dead 0.02 0.09 0.04 -0.01 0.13 0.10 0.00 -0.03 0.02 0.02 0.66 0.55 -0.56 0.70 0.10 -0.42 -0.12 0.04 0.42 inact Dead act 0.15 0.21 0.22 -0.12 0.33 -0.02 0.00 -0.05 0.06 0.01 0.54 0.68 -0.64 0.40 -0.11 -0.46 0.18 -0.30 0.28 0.26 Live inact 0.12 0.07 0.19 0.32 0.38 -0.08 -0.18 0.19 0.12 0.19 0.30 0.46 -0.46 0.31 -0.37 -0.37 0.33 -0.53 0.04 0.26 0.54 Live act -0.10 -0.16 -0.16 -0.11 -0.37 -0.08 0.01 -0.12 -0.15 -0.16 -0.67 -0.73 0.71 -0.69 0.08 0.58 -0.15 0.21 -0.49 -0.82 -0.55 -0.67 168h VAP VSL VCL ALH BCF STR LIN MOT PROG RAPID Dead Morib Live PI+PNA- PI+PNA+ PI- PI- HDS DFI Dead Dead Live PNA- PNA+ inact act inact VAP VSL 0.60 VCL 0.98 0.59 ALH 0.79 0.15 0.78 BCF -0.42 -0.21 -0.36 -0.44 STR -0.67 0.12 -0.67 -0.80 0.27 LIN -0.58 0.18 -0.61 -0.75 0.13 0.93 MOT 0.54 0.35 0.48 0.47 -0.23 -0.33 -0.22 PROG 0.49 0.65 0.44 0.26 -0.21 0.00 0.08 0.87 RAPID 0.73 0.49 0.68 0.59 -0.36 -0.43 -0.32 0.95 0.86 Dead 0.20 0.22 0.17 0.03 -0.22 -0.14 -0.10 -0.06 -0.01 0.02 Morib 0.06 0.05 0.05 0.01 0.12 -0.16 -0.19 -0.09 -0.08 -0.06 0.69 Live -0.05 -0.32 -0.05 0.10 -0.04 -0.11 -0.06 -0.02 -0.14 -0.05 -0.71 -0.66 PI+PNA- 0.13 0.22 0.15 0.00 -0.04 -0.08 -0.13 -0.01 0.04 0.04 0.74 0.61 -0.88 PI+PNA+ -0.03 0.01 -0.07 -0.08 -0.09 0.24 0.20 -0.13 -0.03 -0.11 0.12 0.02 0.16 -0.21 PI-PNA- -0.29 -0.44 -0.25 0.00 -0.11 0.01 -0.01 -0.19 -0.28 -0.24 -0.55 -0.58 0.57 -0.45 -0.25 PI-PNA+ 0.33 0.43 0.28 0.12 0.04 -0.05 -0.01 0.29 0.37 0.33 0.03 0.20 -0.05 -0.11 0.08 -0.64 HDS -0.23 -0.21 -0.22 -0.22 0.11 0.12 0.07 -0.08 -0.11 -0.14 0.07 0.03 0.03 0.09 0.32 -0.17 0.10 DFI -0.12 -0.06 -0.09 -0.07 0.10 -0.04 -0.12 -0.25 -0.23 -0.25 0.52 0.54 -0.56 0.71 -0.08 -0.32 -0.11 0.36 Dead 0.23 0.32 0.22 0.12 -0.01 -0.01 -0.04 -0.01 0.09 0.06 0.66 0.58 -0.60 0.61 0.25 -0.42 -0.09 0.03 0.43 inact Dead act -0.03 -0.13 -0.03 0.02 0.16 -0.24 -0.24 -0.17 -0.20 -0.15 0.35 0.55 -0.34 0.29 -0.17 -0.35 0.14 -0.22 0.50 0.19 Live inact -0.06 0.10 -0.01 -0.21 0.30 0.07 0.03 -0.17 -0.02 -0.15 0.21 0.36 -0.51 0.39 -0.17 -0.11 -0.11 0.00 0.41 0.17 0.32 Live act -0.16 -0.34 -0.17 0.00 -0.13 0.00 0.03 -0.05 -0.19 -0.10 -0.63 -0.71 0.83 -0.71 0.02 0.48 -0.01 0.10 -0.50 -0.76 -0.44 -0.67 240h VAP VSL VCL ALH BCF STR LIN MOT PROG RAPID Dead Morib Live PI+PNA- PI+PNA+ PI- PI- HDS DFI Dead Dead Live PNA- PNA+ inact act inact VAP VSL 0.55 VCL 0.96 0.52 ALH 0.84 0.25 0.86 BCF 0.00 0.47 0.10 -0.12 STR -0.38 0.38 -0.36 -0.44 0.61 LIN -0.33 0.41 -0.37 -0.43 0.51 0.96 MOT 0.83 0.47 0.77 0.65 0.17 -0.18 -0.14 PROG 0.76 0.67 0.72 0.55 0.31 0.05 0.06 0.92 RAPID 0.87 0.52 0.81 0.67 0.15 -0.22 -0.17 0.98 0.92 Dead 0.21 0.09 0.23 0.23 0.11 -0.10 -0.08 0.20 0.13 0.23 Morib 0.15 0.04 0.19 0.12 0.05 -0.06 -0.06 0.16 0.08 0.14 0.66 Live -0.02 0.02 0.01 -0.06 0.05 0.06 0.02 0.03 0.11 0.01 -0.87 -0.57 PI+PNA- -0.09 -0.33 -0.13 0.05 -0.28 -0.34 -0.28 0.02 -0.14 0.02 0.52 0.29 -0.60 PI+PNA+ -0.10 -0.04 -0.05 0.02 -0.15 0.08 0.00 -0.15 0.00 -0.16 0.19 0.42 -0.15 0.20 PI-PNA- -0.22 -0.17 -0.26 -0.15 -0.24 0.06 0.07 -0.34 -0.27 -0.30 -0.77 -0.75 0.65 -0.45 -0.21 PI-PNA+ 0.41 0.35 0.46 0.26 0.37 -0.03 -0.04 0.46 0.38 0.43 0.37 0.29 -0.22 -0.19 -0.43 -0.55 HDS 0.19 0.16 0.21 0.14 0.15 -0.02 -0.03 0.20 0.27 0.19 -0.04 -0.26 0.30 -0.06 -0.11 0.11 -0.05 DFI 0.07 -0.02 -0.01 0.01 -0.29 -0.20 -0.08 0.19 0.07 0.21 0.24 0.30 -0.22 0.49 0.24 -0.22 -0.19 -0.06 Dead 0.09 -0.17 0.08 0.21 -0.48 -0.33 -0.27 0.07 -0.07 0.11 0.40 0.51 -0.37 0.56 0.43 -0.32 -0.27 -0.12 0.64 inact Dead act -0.34 -0.47 -0.38 -0.26 -0.25 -0.19 -0.18 -0.23 -0.32 -0.27 0.14 0.35 -0.26 0.17 0.26 -0.12 -0.08 -0.56 0.31 0.11 Live inact -0.13 0.13 -0.11 -0.22 0.27 0.38 0.40 -0.08 -0.03 -0.08 0.32 0.40 -0.43 -0.04 -0.05 -0.31 0.29 -0.38 -0.08 0.04 0.05 Live act 0.12 0.12 0.13 0.13 0.20 0.07 0.04 0.06 0.13 0.04 -0.53 -0.71 0.60 -0.39 -0.33 0.54 -0.03 0.42 -0.49 -0.73 -0.36 -0.54 Fig. 3 Spearman’s correlation coefficients between all analyzed sperm parameters in boar spermatozoa stored for 240 h at 17 °C (n 35). Values in = bold font—statistically significant correlation (P < 0.05). VAP-average path velocity; VSL-straight line velocity; VCL-curvilinear velocity; ALH-amplitude of lateral head displacement; BCF-beat cross frequency; STR-straightness; LIN-linearity; mot-percentage of motile spermatozoa; PMOT-percentage of progressively motile spermatozoa; RAPID-subpopulation of rapid cells; Live—SYBR PI; PI PNA live with intact acrosome; HDS—%HDS (high + − − DNA stainability) the percentage of spermatozoa with immature chromatin; DFI—%DFI (DNA fragmentation index) percentage of spermatozoa with DNA fragmentation; Live act—live with active mitochondria (Rhodamine PI ) + − Bielas et al. Acta Vet Scand (2017) 59:58 Page 9 of 12 previous observations about relatively low levels of %DFI sperm viability. It can be assumed that these slight differ- in fresh boar semen [16, 26, 31, 34]. ences may have no biological significance. This assump- It has been proved that differences in sperm DNA tion supports the conclusion presented by Hernandez damage between ejaculates can result from external fac- et al. [38] who stated that the low overall DNA damage tors such as collection procedure, handling, dilution or observed in frozen-thawed spermatozoa seemed to have internal factors, e.g., inherent chromatin packaging of the little biological importance. Waberski et  al. [16] also spermatozoa, the composition of seminal plasma and of demonstrated that evaluation of sperm chromatin struc- accessory gland fluid including zinc ions, zinc-binding tural integrity by the SCSA has only limited value for proteins, low molecular weight antioxidants and proteins identifying deficiencies in normospermic fresh or stored with antiperoxidant properties [16, 34]. There is a current boar spermatozoa. debate about whether these intrinsic or extrinsic factors The values of %HDS in boars of the Polish White Large cause different reactions of the sperm chromatin to the breed were similar to values obtained by others [12] for SCSA procedure. In our study, the influences of external Landrace, Danish Large White and Hampshire boars. factors were minimized just as in earlier studies [16, 34]. We did not observe any trend of decrease or increase of However, in the present study, sperm rich fractions were %HDS during sperm storage. The similarity of HDS val- used, which may mean that the antioxidant properties of ues during the whole period of semen storage is probably the entire seminal plasma were reduced [51]. due to the fact that the %HDS value is mainly determined In the present study, the average %DFI of spermatozoa by the initial integrity of DNA structure resulting from in fresh semen after extension (0 h) in 35 ejaculates from the quality of spermatogenesis. In humans, the popula- seven boars was 3.55%. We found an initial significant, tion of %HDS is supposedly composed of immature cells abrupt rise of %DFI at 0 h and again at 48 h of incuba- that lack chromatin condensation [52] and may also rep- tion in long-term extender. The %DFI obtained here resent doublets or triplets of spermatozoa. This is con- increased slightly but significantly during the 240 h of sistent with our observation that storage of boar semen storage after collection, up to an average of 4.71   2.2%. did not increase the %HDS population. Thus, it seems ± This increase was greatest ( 34.48%) between day 0 and that %HDS is not a useful marker of changes in the qual- + day 2, and in most boars the percentages of spermatozoa ity of liquid stored boar spermatozoa. with fragmented DNA were almost always lower than 5% Significant differences in chromatin structure of up to 240 h of storage. Our data agree with the results stored spermatozoa between individual boars were of Broekhuijse et al. [34] who reported that the %DFI at also detected. The study included only seven boars, but day 0 was 3.15% and increased to 4.19% during 15 days there were differences among males with respect to of liquid storage, and the greatest increase of %DFI was the quality of their sperm characteristics during stor- observed between day 0 and day 1. age. We discovered a significant influence of boar on It was also previously reported that extended boar chromatin integrity (Table  2) in spermatozoa stored spermatozoa showed an increase in DNA instability from for 240 h at 17 °C, and also on the sperm motion char- day 0 to day 4 in some extenders [12, 28]. Contrary to acteristics (with the exception of BCF) (Table 1), on this, De Ambrogi et al. [19] suggested that the customary plasma membrane integrity (Table  2), on acrosome storage of boar semen for 96 h at 17 °C was too short an integrity (Table 2) and on mitochondrial activity (with interval to cause loss of integrity in nuclear DNA. Similar the exception of dead sperm cells with active mitochon- results were obtained by Waberski et al. [16] in the first dria) (Table 2). These study reveal that there is individ- part of their study. However, in the second part of their ual variation among boars concerning preservation of study, they obtained a slight but significant increase in DNA integrity during storage, which is in accordance mean %DFI results from 2.2% initially up to 2.7% at 120 h with Fraser and Strzeżek [25] and Sutkeviciene et al. of semen storage. [53]. This indicates that the effect of individual boar is In our study, only two out of 35 ejaculates showed an of great importance concerning sperm quality during increase of %DFI above 10% (12.6; 10.8) during 240 h longtime storage and must always be taken into account of storage. This is in accordance with findings of other in the assessment both of DNA fragmentation and other authors who showed a significant increase of DFI during sperm variables. No previous studies have investigated 168 h of storage in only three out of 42 ejaculates [16]. sperm DNA integrity using the SCSA parameters in the The results support the concept of relatively low sen- semen of normospermic, healthy boars of the Polish sitivity of boar sperm DNA to defragmentation during Large White breed. However, our preliminary results storage of liquid semen [12, 16, 19, 34]. The differences need to be investigated further in a larger study to eval- in DNA fragmentation on different days of storage were uate and understand the precise mechanism maintain- generally rather low compared to changes in motility or ing sperm DNA integrity. Bielas et al. Acta Vet Scand (2017) 59:58 Page 10 of 12 Sperm characteristics, especially all motility and struc- for proper evaluation of motion properties of stored tural parameters, were also significantly affected by the boar spermatozoa. It should be borne in mind that we storage time. This means that the time points when these revealed almost no correlation between motility fea- characteristics were assessed during storage had a signifi- tures and structural sperm parameters. The high cor- cant impact on these properties of the stored boar sperm relation between all motility parameters and the lack of cells. correlation between motility and structural features was The interaction of boar and time of incubation as a characteristic during the whole time of sperm storage. source of variability only affected motility parameters Therefore, motility and structure may be treated as sepa- (without MOT) assessed by CASA during storage, which rate, partly independent features that always have to be indicates that the boar factor as well as time of semen separately assessed. storage play important roles in assessment of these motil- We did not observe abrupt changes of mitochondrial ity traits during long-term liquid storage of boar semen. activity over time in the populations of live and dead Meanwhile, the non-significant interactions between spermatozoa. It is obvious, that in the population of boar and time points in the remaining assays of sperm dead spermatozoa the proportion of cells with inactive characteristics indicate that influences of boar and time mitochondria remained nearly unchanged at consecu- of semen storage were homogeneously distributed among tive assessment points. However, it is more difficult to the sperm parameters studied. This may mean that these understand why changes in mitochondrial potential were characteristics of spermatozoa provide an additive value so subtle in the group of live cells in spite of the rapid in assessing the quality of long-term liquid-stored boar decrease in progressively motile spermatozoa. It appears spermatozoa. that the dynamics of the increase in percentages of live Significant negative correlations among SCSA variables cells with inactive mitochondria were similar to the and most classical sperm quality parameters in fresh and dynamics of other tests performed on the flow cytometer cryopreserved semen were shown in rams [54], bulls [55] rather than the dynamics of motility changes recorded on and humans [22]. The significant correlations between CASA. SCSA and classical sperm quality parameters suggest Conclusions that, taken together, both types of assays are better pre- dictors of sperm quality and male fertility than each one The most sensitive method for assessing changes in separately. The negative correlation between %DFI and sperm cell features during storage at 17 °C are those sperm viability was also detected in stored boar sperma- describing populations of motile cells and parameters tozoa [12]. In another study [19] when ejaculates from related to speed of motility. Plasma and acrosome mem- only four boars were included, the increase of %DFI was brane integrity and mitochondrial function charac- accompanied by increased deterioration of sperm plasma teristics are relatively resistant to storage in long-term membrane integrity during storage. Other researchers semen extender and change to a lesser degree. Although [34] did not find a significant correlation between the increased DNA fragmentation was revealed, the extent %DFI and the standard boar sperm variables during long- of these changes was relatively low and it appears that term storage. We found significant correlations between extenders efficiently protect DNA structure. These find- %DFI and %HDS and structural sperm parameters at ings support the opinion that the SCSA test has rela- 0 h of storage after which the only significant correla- tively little value for routine evaluation of changes in boar tions were observed for %DFI. It may be concluded that sperm characteristics during semen storage in long-term %DFI changes are associated with the disruption of other extender. sperm structures during storage. On the other hand, Authors’ contributions %HDS is the parameter associated with abnormalities WB participated in the study design and was involved in manuscript prepara- of spermatogenesis and is only partly independent from tion; WN contributed to the study design, interpretation of the results, coordi- nated clinical work and was responsible for revision of the manuscript; AP per- storage time. Correlations between both parameters and formed semen analysis, and participated in statistical analysis; AR participated structural features were lost at 240 h of storage. This in collection of samples and coordinated clinical work; RM participated in the may indicate that assessing DNA integrity has an addi- collection of samples. All authors read and approved the final manuscript. tive value for standard sperm assessment only in cases of Author details extremely long storage times. 1 Department of Reproduction and Clinic of Farm Animals, Faculty of Vet- It is noteworthy that detailed analysis of correlations erinary Medicine, Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 49, 50-366 Wrocław, Poland. 2 Department of Immunology, between values obtained in the present study revealed Pathophysiology and Veterinary Prevention Medicine, Faculty of Veterinary high, significant correlations among the majority of Medicine, Wrocław University of Environmental and Life Sciences, ul. C. K. motility parameters. Therefore, it may be suggested that Norwida 31, 50-375 Wrocław, Poland. 3 Department of Internal Medicine and Clinic of Diseases of Horses, Dogs and Cats, Faculty of Veterinary Medicine, routine analysis on 1–2 motility parameters is adequate