COMPARATIVE STUDY OF DIFFERENT METHODS FOR PLASMA MEMBRANE INTEGRITY ASSESSMENT OF FROZEN-THAWED BOAR SPERMATOZOA

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1 Bull Vet Inst Pulawy 55, , 211 COMPARATIVE STUDY OF DIFFERENT METHODS FOR PLASMA MEMBRANE INTEGRITY ASSESSMENT OF FROZEN-THAWED BOAR SPERMATOZOA LEYLAND FRASER, ŁUKASZ ZASIADCZYK, MAREK LECEWICZ, AND WŁADYSŁAW KORDAN Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, Warmia and Mazury University in Olsztyn, 1-71 Olsztyn, Poland, Received: May 12, 21 Accepted: February 2, 211 Abstract In this study, different assays were used to assess the structural and functional integrity of the sperm plasma membrane following freezing-thawing of the whole ejaculates (WEs) and sperm-rich fractions (SRFs) of boar semen. Besides sperm viability assessments (motility and mitochondrial membrane potential using a mitochondrial specific dye, JC-1 with propidium iodide, PI), sperm plasma membrane integrity (PMI) assessments were determined simultaneously using different membrane-based tests (SYBR- 1/PI and carboxyfluorescein diacetate (CFDA) with PI (CFDA/PI), and the hypo-osmotic swelling (HOS) test). ANOVA results showed that boar variability had a significant effect on the analysed parameters of post-thaw sperm characteristics. Spermatozoa harvested from the WEs exhibited a marked decline in post-thaw viability, manifested in reduced motility and mitochondrial membrane potential, than those originated from the SRFs after freezing-thawing. Cryopreservation compromised sperm PMI, as indicated in a significant decline in SYBR-stained, CFDA-stained or HOS-positive sperm cells, irrespective of the ejaculate collection procedure. It was observed that the membrane-based tests were strongly inter-correlated. Furthermore, agreement between the measurements of the membrane-based tests was confirmed by the Bland-Altman scatter plots of differences, suggesting that these tests could detect the same sperm cohorts, which were susceptible to cryo-induced membrane damage. The findings of this study indicate that dual fluorescent staining with SYBR-1/PI and CFDA/PI assays, in combination with the HOS test, provide more precise description of the sperm populations in frozen-thawed boar semen. Key words: boar, spermatozoa, plasma membrane integrity, cryopreservation. The structural and functional integrity of the plasma membrane integrity of spermatozoa is one of the most important aspects of sperm biology (, 12). An intact sperm plasma membrane is a prerequisite for normal cell function and several events associated with egg-sperm fertilisation (1, 17). Evaluation of the sperm plasma membrane integrity (PMI) is a key step in the process of development of new cryopreservation protocols because these sperm membrane structures are highly susceptible to cryo-induced oxidative damage (1). A plethora of fluorescent-based dyes, such as DNA-binding probes SYBR-1, Hoechst 3325, propidium iodide (PI), ethidium homodimer, and esterase-bound probes, such as carboxyflourescein diacetate (CFDA), have been used with microscopic techniques to monitor sperm PMI in liquid-stored or frozen-thawed boar spermatozoa (, 5, 7, 1, 11). These membrane-impermeable fluorescent dyes are based on exclusion principle, using the rationale that spermatozoa that can exclude these dyes are viable. A similar rationale lies behind the hypo-osmotic swelling (HOS) test, in which spermatozoa with functional intact membranes are able to react to a hypo-osmotic environment (). Molecular changes in the sperm membrane structures, incurred during the freezing-thawing process, are associated with reduced post-thaw sperm survival (2, 13). During freezing-thawing spermatozoa are exposed to non-physiological concentrations of extracellular solutions as well as the potential of lysis due to ice crystal formation, resulting in physical damage to their morphological structure (9). Boar sperm plasma membrane consists of specialised domains, which are exceptionally sensitive to cryo-induced oxidative stress (2). Sperm injury is manifested in a loss of selective permeability of the plasma membrane, outer acrosomal membrane and impaired metabolic activity (13). Moreover, fluorescent probes are used to assess differing degrees of subtle cellular damage that spermatozoa would undergo during the freezing-thawing process (9, 11, 1). The relevance of detecting subtle changes in the sperm plasma membrane has been emphasised by the findings of Juonala et al. (1) and Sutkeviciene et al. (17), which demonstrated that sperm PMI in liquid-stored boar semen was significantly correlated with fertility parameters such as nonreturn rate and litter size after artificial inseminations in sows.

2 232 It should be emphasised that the capacity of boar spermatozoa to withstand freezing-thawing process depends on their membrane fluidity, which could be influenced by the presence of seminal plasma components (6). This study was designed to find out whether different membrane-based tests could detect the same sperm populations with damaged membrane structures following freezing-thawing of spermatozoa from the whole ejaculates (WEs) and sperm-rich fractions (SRFs) of boar semen. Besides assessment of the sperm PMI (SYBR-1/PI and CFDI/ PI assays) and the hypo-osmotic swelling (HOS) test, sperm motility and mitochondrial membrane potential (JC-1/PI assay) were also evaluated. Material and Methods Ejaculate collections. Semen was collected from three Polish Large White boars, designated A, B, and C (aged 2 years), once weekly for a period of 5 weeks. The boars were fed a commercial porcine ration. Water was available ad libitum. Semen was collected using the gloved-hand technique, and the gel portion was removed using nylon mesh filter. Sperm concentration was calculated with a haemocytometer. The percentage of morphologically abnormal spermatozoa was evaluated using the Giemsa staining method. Permission to conduct this study was granted by the Local Ethics Committee Collections of the whole ejaculates (WEs) and only the sperm-rich fractions (SRFs) were performed after weekly rotation regimens. The WEs were collected on the first week followed by the collection of the SRFs from the same boars on the subsequent week. This protocol of semen collection was repeated for a period of 6 successive-weeks, giving a total number of nine WEs and nine SRFs, three from each boar, respectively. Semen samples with at least 7% motile spermatozoa and more than 5% morphologically normal spermatozoa were used. Sperm concentration was assessed cytometrically, using a Bűrker chamber. Spermatozoa were assessed for motility, plasma membrane integrity, and mitochondrial function prior to, and after freezing-thawing, as described in a previous study (5). Semen cryopreservation. Ejaculates were processed according to a standard protocol (15), with modifications (5). Lipoprotein fraction was isolated from ostrich egg yolk (LPFo) and lyophilised until required (16). Semen was diluted with a standard boar semen extender, Kortowo 3 (K3), cooled for 3 h at 16 C, and then centrifuged ( g for 1 min). The sperm pellets were re-suspended in a cooling extender containing 11% lactose with LPFo and cooled for 3 h at 5 C. The cooled sperm samples were further diluted (2:1) with a freezing extender containing 9.5 ml of lactose-lpfo extender, 9 ml of glycerol (v/v), and 1.5ml of Orvus Es Paste (lactose-lpfo-g). The final concentrations of LPFo, glycerol, and Orvus Es Paste were 5%, 3%, and.5%, respectively. All samples were packaged in 1-mL sterilised aluminium tubes and frozen in a controlled programmable freezer (Ice Cube 11, SY-LAB, Austria). The tubes were plunged into liquid nitrogen and stored until required. Prior to sperm quality analysis, the tubes were thawed in a water bath for 6 s at 5 C. Aliquots of sperm samples were diluted with K3 extender (3 1 6 spermatozoa/ml) at 37 C prior to assessment. Sperm viability assessment Motility evaluations. Sperm motility was evaluated by placing 6 L of sperm sample on prewarmed microscope slide and examined at 2 magnification under a light microscope (Olympus CH 3, Japan) equipped with a heated stage (37 C). Mitochondrial membrane potential (MMP). Mitochondrial energy status in spermatozoa was assessed in aliquots of diluted semen, using a fluorescent probe, 5,5,6,6 -tetrachloro-1,1,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Molecular Probes, USA), according to a previously described method (3). Aliquots (1 µl) of stained sperm samples were examined under an epifluorescence microscope (Olympus CH 3, Japan). Sperm cells displaying only orange-red fluorescence at the mid-piece region were considered viable spermatozoa with functional high MMP. Two slides were evaluated per sample and 2 spermatozoa were counted per slide. Assessment of sperm plasma membrane integrity (PMI). Sperm plasma membrane integrity was assessed with dual fluorescent probes, SYBR-1 and PI (Live/Dead Sperm Viability Kit; Molecular Probes, USA), according to a previous method (6). Aliquots (1 µl) of stained sperm cells were placed on microscopic slides, covered with cover slips and examined at 6 magnification under epifluorescence microscopy (Olympus CH 3). Sperm cells displaying only bright green fluorescence in the head region were considered viable spermatozoa with intact membrane. Two slides were evaluated per sample and 2 spermatozoa were counted per slide. Dual fluorescent staining technique was used to assess sperm plasma membrane integrity (), using CFDA ( mg/ml DMSO) with PI (.5 mg/ml PBS). Aliquots (1 µl) of samples stained with CFDA/PI were examined under an epifluorescence microscope (Olympus CH 3). Sperm cells displaying only bright green fluorescence throughout the entire surface were considered viable spermatozoa with intact membrane. A minimum of 2 cells per slide was examined in random fields for each aliquot. The HOS test was conducted as previously described (). Briefly, aliquots (1 µl) of semen samples were mixed with 1 ml of hypo-osmotic solution (15 mosm/kg) prepared with 75 mmol/l fructose and 25 mmol/l sodium citrate and incubated at 37 C for 6 min. After incubation, the sperm suspensions were thoroughly mixed, a 1 µl drop was placed on a microscopic slide, covered with a coverslip, and examined at magnification under phase-contrast microscope. Spermatozoa with intact plasma membranes displayed swollen tails in response to the

3 233 treatment. For each membrane integrity assay, two slides were evaluated per sample and 2 spermatozoa were counted per slide. Statistical analysis. All results are expressed as mean ± standard error of the mean (S.E.M). A total of five ejaculates, each from three boars, were examined by repeated measures ANOVA using general linear model (GLM) procedure from Statistica software package, version 9 (StatSoft Incorporation, USA). The statistical model included the effects of boar and ejaculate collection procedure (3 2) and their interactions on sperm viability and plasma membrane integrity. Correlations were performed using the Pearson correlation test. Significant main effects and the interaction means were compared using the Neuman- Keuls post hoc test (P<.5). The scatter plot of differences between the measurements of the membrane integrity assays (y-axis) were plotted against the average (bias) measurements (x-axis) with the 95% confidence interval (CI) limits to study the agreement between the membrane integrity tests, using the method of Bland and Altman (1). Results There were no marked differences among the boars in any of the analysed sperm parameters between samples from the WEs and SRFs before freezingthawing. The results of sperm motility ranged from 71.5% to 7.5% with a mean of 7. ±.% (mean ± S.E.M). Sperm MMP ranged from 3.6% to 6.2%, with a mean of.3 ±1.2%. Furthermore, sperm PMI assessed by SYBR-1/PI assay averaged 77. ±1.% (range, 75.% to 6.%), whereas with CFDA/PI assay the mean was 7.5 ±.9% (range, 76% to 2.6%). The values for HOS test averaged.7 ±.9% (range,.% to 5.6%). The results of ANOVA revealed that boar variability had a significant effect (P<.1) on the analysed sperm characteristics following freezingthawing. It was observed that the ejaculate collection procedure (WE and SRF) was the only source of variation exerting a significant effect on post-thaw sperm motility (P<.13) and MMP (P<.32). Table 1 shows post-thaw sperm quality characteristics in the WEs and SRFs. It was observed that spermatozoa harvested from the WEs exhibited a marked decline (P<.5) in post-thaw viability, manifested in reduced motility and MMP, assessed by the JC-1/PI assay, compared with those originated from the SRFs. Differences in post thaw sperm motility and MMP among boars were evident, being significantly lower in boar A compared with boar C. Post-thaw sperm PMI, assessed by SYBR-1 assay, CFDA/PI assay or HOS test, was not significantly differed (P>.5) from the WEs and SRFs. There were differences in post-thaw sperm PMI between WEs and SRFs within boars. Spermatozoa of boar C exhibited significantly higher (P<.5) post-thaw PMI. The agreement between post-thaw sperm PMI, assessed with different membrane integrity tests, as obtained from the scatter plots of differences of the measurements, is shown in Fig. 1. It seemed that the distributions of the data points for the membrane integrity assays were random and the scatter of most of the data points were within or close to the 95% confidence intervals for the limits of agreement, irrespective of the ejaculate collection procedure. The average percentage bias for the membrane integrity assay comparisons was around the zero line. However, CFDA/PI assay gave higher estimates in the percentage of spermatozoa with intact membrane compared with the SYBR-1/PI assay (Figs 1A and B). A similar trend was shown for the HOS test when compared with SYBR- 1/PI assay (Figs 1C and D) and CFDA/PI assay (Figs 1E and F). Post-thaw sperm mitochondrial function, measured with JC-1/PI assay, was significantly correlated with the percentage of motility in sperm samples of the WE (r =.5; P<.5) and SRF (r =.53, P<.5). It was observed that values of JC-1/PI assays were positively correlated with either the values of SYBR-1/PI and CFDA/PI assays or HOS test (Table 2). Furthermore, measurements of the different membrane integrity tests, SYBR-1/PI and CFDA/PI assays and HOS test, were significantly inter-correlated (Table 2). Discussion In the current study post-thaw sperm viability, expressed by motility and MMP, and the plasma membrane integrity, assessed by different assays, were evaluated in the WEs and SRFs of individual boars. The results showed that increased cryo-induced damage to the sperm membrane structures was concurrent with impaired sperm motility and MMP. We propose that freezing-thawing had different effects on post-thaw sperm characteristics, indicating that different measures of sperm function can respond independently to cryoinduced oxidative stress. The spermatozoon is a complicated cell, having multiple cell compartments (12). In the current study the use of different methods to assess multiple cell attributes provide more descriptions of the sperm populations following freezing-thawing. The findings of the present study showed that significant differences in post-thaw sperm viability between semen samples harvested from the WEs and SRFs were manifested by higher proportions of motile spermatozoa with functional mitochondria in the SRFs. Moreover, the interactions of spermatozoa with cryoprotective components of the extender during cooling and freezing appear to reduce damage to their membrane structures, thereby maintaining the sperm functions, such as viability and PMI (3, 5, 9, 16). However, it should be noted that the beneficial effects of boar seminal plasma on post-thaw sperm function is hard to define due to its complexity (5). Evaluation of the post-thaw sperm PMI using the SYBR-1/PI assay, CFDA/PI assay or the HOS test did not reveal significant differences between the WEs and SRFs.

4 23 Table 1 Sperm viability and plasma membrane integrity in the whole ejaculate (WE) and sperm-rich fraction (SRF) of boar semen following freezing-thawing WE SRF Boar Sperm viability (%) Sperm PMI (%) Sperm MF (%) Sperm PMI (%) Motility MMP SYBR-1/PI CFDA/PI HOS test Motility MMP SYBR-1/PI CFDA/PI HOS test A 2.5 ± 1.5 a,xy 35.6 ± 2.3 a,xy B 22. ± 1.9 a,x 31.6 ± 1.5 a,x 37.2 ± 1.6 a,x 39.2 ± 2.6 a,xy.2 ± 3.5 a,xy 3. ± 2.3 b,xy. ± 1.9 a,x 39.3 ± 1.1 a,x 1. ± 1. a,xy.5 ± 3.2 a,xy 3.3 ± 1.6 a,x 33. ± 1.9 a,x 3. ± 2.1 a,x 2.5 ± 1.2 b,x 39.7 ± 1.7 b,x 35.2 ± 2.1 a,x 37. ± 1.3 a,x.2 ± 3.1 a,x C 3.3 ± 1.5 a,y 2.7 ± 1.1 a,y.6 ± 1.9 a,y 5.2 ±1.7 a,y 9.2 ± 2.5 a,y 37.5 ± 1.7 b,y 5.9 ± 2.1 b,y 51. ± 1.3 a,y 9.2 ± 2.2 a,y 53. ± 3.7 a,y MMP sperm mitochondrial membrane potential assessed with JC-1 and propidium iodide (JC-1/PI assay), SPMI sperm plasma membrane integrity simultaneously assessed with the SYBR-1/PI assay, 6-carboxyofluorescein acetate and PI (CFDA/PI assay) and hypo-osmotic swelling (HOS) test. Values represent the means (± S.E.M) of five ejaculates, each from three boars. Within analysed parameter (WE vs SRF), values with different superscripts (a,b) are significant at P<.5 (Neuman-Keuls post hoc test), within columns, values with different superscripts (x, y) are significant at P<.5 (Neuman-Keuls post hoc test). Table 2 Correlation coefficients of post-thaw sperm characteristics in the whole ejaculate (WE) and sperm-rich fraction (SRF) of boar semen following freezing-thawing Parameters WE SYBR-1/PI CFDA/PI HOS test SYBR-1/PI CFDA/PI HOS test SRF JC-1/PI.92***.96***.9***.91***.9***.9*** SYBR-1/PI -.95***.93*** -.9***.9*** CFDA/PI *** - -.9*** *** P<.1.

5 Difference of CFDA/PI assay and HOS test (%) Difference of CFDA/P Iassay and HOS test (%) Difference of SYBR-1/PI assay and HOS test (%) Difference of SYBR-1/PI assay and HOS test (%) Difference of SYBR-1/PI and CFDA/PIassay (%) Difference of SYBR-1/PI and CFDA/PIassay (%) (A) 12 (B) Avearage of SYBR-1/PI and CFDA/PI assay (%) 12 (C) Avearage of SYBR-1/PI and CFDA/PI assay (%) 12 (D) (E) Avearage of SYBR-1/PI assay and HOS test (%) Avearage of SYBR-1/PI assay and HOS test (%) 12 (F) Avearage of CFDA/PI assay and HOS test (%) Avearage of CFDA/PI assay and HOS test (%) Fig. 1. Scatter plots of differences (n=3) for sperm plasma membrane integrity in the whole ejaculate (WE) and spermrich fraction (SRF) of boar semen following freezing-thawing. Measurement comparisons were performed for the SYBR-1/PI and CFDA/PI assays in the WE (A) and SRF (B); SYBR-1/PI assay and the hypo-osmotic swelling (HOS) in the WE (C) and SRF (D); CFDA/PI assay and the HOS test in the WE (E) and SRF (F). The scatter plots of differences between the measurements of the membrane integrity assays (y-axis) were plotted against their average measurements (x-axis). The average bias is indicated by the solid line and the 95% confidence intervals for the limits of agreement are represented by the broken lines. The fluorescent probes, such as SYBR-1, PI and CFDA, measure structural damage to plasma membrane of spermatozoa (, 6, 7, 12). Even though the mechanism overlying the staining principle of the DNAspecific probe, SYBR-1 and the esterase-based dye, CFDA, are different, the findings of the current study showed that they detected similar sub-sperm populations, which were susceptible to cryo-induced damage. Furthermore, membrane changes related to cryo-induced injury of spermatozoa, as assessed by the fluorescent-based dyes, were reflected in the proportions of HOS-negative frozen-thawed spermatozoa. The HOS test, which measures functional changes in the sperm plasma membrane structures, reflects the activity required by the membrane to establish osmotic equilibrium (). Furthermore, the Bland-Altman plot of differences showed that the measurements of SYBR- 1/PI assay, CFDA/PI assay, and the HOS test yielded acceptable agreement, suggesting that these tests could detect the same sperm cohorts with compromised PMI

6 236 after freezing-thawing. Evidence has been shown that the Bland-Altman scatter plots constitute an appropriate technique used to ascertain reliability of comparison agreement between two variables (1). It can be suggested that the Bland-Altman scatter plots, in conjunction with the correlation analysis, which measures the strength of the relationship between the SYBR-1/PI assay, CFDA/PI assay, and the HOS test, provides reliable information about the extent of the agreement between the measurements of the membranebased tests. The use of the different membrane-based tests to assess post-thaw sperm PMI revealed that there was variability among boars to respond to cryopreservation, regardless of the ejaculate collection procedure. Moreover, individual differences between boars in the ability of spermatozoa to withstand cryopreservation may be attributed to intrinsic genetic traits (1). Additionally, it is important to emphasise that male-tomale differences in cryo-tolerance might be contributing factors to the variations in sperm viability and plasma membrane integrity. Simultaneous application of a combination of fluorescent probes allows the detection of subtle changes in the sperm membrane structures following freezingthawing, providing more information about the nature of the cryo-induced injury of spermatozoa at different subcellular regions. It can be concluded that the use of different fluorescent probes, in conjunction with the HOS test, allows for better discrimination among ejaculates of boars following freezing-thawing and may help to predict the freezability of boar semen. Acknowledgments: This study was supported by research funds (No ) from the University of Warmia and Mazury in Olsztyn. References 1. Bland J.M., Altman D.G.: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 196, 1(76), Buhr M.M., Curtis E.F., Somnapan Kakuda N.: Composition and behaviour of head membrane lipids of fresh and cryopreserved boar sperm. Cryobiology 199, 31, Dziekońska A., Fraser L., Strzeżek J.: Effect of different storage temperatures on the metabolic activity of spermatozoa following liquid storage of boar semen. J Anim Feed Sci 29, 1, Fraser L., Lecewicz M., Strzeżek J.: Fluorometric assessments of viability and mitochondrial status of boar spermatozoa following liquid storage. Pol J Vet Sci 22, 5, Fraser L., Strzeżek J.: Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezingthawing. Anim Reprod Sci 27, 99, Garner D.L., Johnson L.A.: Viability assessment of mammalian sperm using SYBR-1 and propidium iodide. Biol Reprod 1995, 53, Gączarzewicz D., Piasecka M., Udała J., Błaszczyk B., Stankiewicz T., Laszczyńska M.: Plasma membrane changes during the liquid storage of boar spermatozoa: a comparison of methods. Acta Vet Hung 21, 5, Jeyendran R.S., van der Ven H.H., Perez-Pelaez M., Crabo B.G., Zaneveld L.J.D.: Development of an assay to assess the functional integrity of the human sperm plasma membrane and its relationship to other semen characteristics. J Reprod Fertil 19, 7, Johnson L.A., Weitze K.F., Fiser P., Maxwell W.M.: Storage of boar semen. Anim Reprod Sci 2, 62, Juonala T., Salonen E., Nurttila T., Anderson M.: Three fluorescence methods for assessing boar sperm viability. Reprod Dom Anim 1999, 3, Kordan W., Lecewicz M., Tobolski J.: Effect of platelet activating factor on motility, plasmalemma integrity, the process of capacitation and acrosome reaction of fresh and cryopreserved boar spermatozoa. Pol J Vet Sci 29, 12, Moce E., Graham J. K.: In vitro evaluation of sperm quality. Anim Reprod Sci 2, 15, Parks J.E., Lynch D.V.: Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 1992, 29, Peña F.J.: Detecting subtle changes in sperm membranes in veterinary andrology. Asian J Androl 27 9, Strzeżek J., Glogowski J., Hopfer E., Wojtkiewicz K.: The Kortowska method of freezing boar semen. Medycyna Wet 195, 1, Strzeżek J., Lecewicz L., Dziekońska A., Fraser L.: A simple method of extraction of lipoprotein fractions from avian egg yolk protective effect on cooled boar semen. Theriogenology 25, 63, Sutkeviciene N., Riskeviciene V., Januskauskas A., Zilinskas H., Andersson M.: Assessment of sperm quality traits in relation to fertility in boar semen. Acta Vet Scand 29, 51, 53; doi:1. 116/ Thurston L.M., Siggins K., Mileham A.J., Watson P.F., Holt W.V.: Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation. Biol Reprod 22, 66,