SINGLE UV EXCITATION OF HOECHST AND PROPIDIUM IODIDE FOR VIABILITY ASSESSMENT OF RHESUS MONKEY SPERMATOZOA USING FLOW CYTOMETRY

Archives of Andrology Journal of Reproductive Systems ISSN: 0148-5016 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/iaan19 SINGLE UV EXCITATION OF HOECHST 33342 AND PROPIDIUM IODIDE FOR VIABILITY ASSESSMENT OF RHESUS MONKEY SPERMATOZOA USING FLOW CYTOMETRY K. Cai, J. Yang, M. Guan, W. Ji, Y. Li & W. Rens To cite this article: K. Cai, J. Yang, M. Guan, W. Ji, Y. Li & W. Rens (2005) SINGLE UV EXCITATION OF HOECHST 33342 AND PROPIDIUM IODIDE FOR VIABILITY ASSESSMENT OF RHESUS MONKEY SPERMATOZOA USING FLOW CYTOMETRY, Archives of Andrology, 51:5, 371-383, DOI: 10.1080/014850190924485 To link to this article: https://doi.org/10.1080/014850190924485 Published online: 09 Jul 2009. Submit your article to this journal Article views: 71 View related articles Citing articles: 8 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=iaan19 Download by: [46.3.207.126] Date: 30 December 2017, At: 22:59

Archives of Andrology, 51:371 383, 2005 Copyright # Taylor & Francis Inc. ISSN: 0148-5016 print/1521-0375 online DOI: 10.1080/014850190924485 SINGLE UV EXCITATION OF HOECHST 33342 AND PROPIDIUM IODIDE FOR VIABILITY ASSESSMENT OF RHESUS MONKEY SPERMATOZOA USING FLOW CYTOMETRY K. Cai & Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming Yunnan, P.R.China and Graduate School, The Chinese Academy of Sciences, Beijing, P.R.China J. Yang, M. Guan, and W. Ji & Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming Yunnan, P.R.China Y. Li & Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming Yunnan, P.R.China and College of Food Science and Technology, Yunnan Agricultural University, Kunming Yunnan, P.R.China W. Rens & Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge, UK & Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UVexcited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342=PI=SYBR-14 triple staining method, it was found that the live=dead sperm distributions were completely concordant in both H342=PI and SYBR-14=PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better preservation of plasma and acrosome integrity than TEST. Therefore, the H342=PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function. Keywords flow cytometry, fluorescent probes, rhesus monkey, spermatozoa, viability This work was supported by grants from the China National Science Foundation (30370166), Major Basic Research Program (G2000016108), and the Chinese Academy of Sciences (KSCX1-05). Address correspondence to W. Ji, Kunming, Institute of Zoology, The Chinese Academy of Sciences, 32 Eastern Jiaochang Road, Kunming, Yunnan 650223, People s Republic of China. E-mail: wji@mail.kiz.ac.cn

372 K. Cai et al. Semen analysis is of prime importance in the diagnosis of male-origin subfertility and in the assisted reproductive technique. Flow cytometry is the current technical solution for rapid, precisely reproducible assessment of cell suspensions including sperm samples [10]. Using flow cytometric assays, multiple sperm attributes can be measured simultaneously in sperm cells [8]. The integrity of sperm membrane, namely, viability, is fundamental for the fertilizing capacity of spermatozoa. Many fluorescent probes have been used either singularly or in combination to assess sperm viability by flow cytometry, among which the SYBR-14 and propidium iodide (PI) dual staining is the mostly accepted method [4 7, 11, 16]. However, SYBR-14 is a green fluorescent dye, and the commonly used acrosomal probes fluorescein isothiocyanate-conjugated lectins, such as peanut agglutinin (FITC-PNA) [12, 13, 16] or pisum sativum agglutinin (FITC-PSA) [7, 11], also emit green fluorescence; thus, it is not suitable to combine the SYBR-14=PI dual staining with FITC-conjugated lectins for multiparameter analysis. Another vital DNA stain Hoechst 33342 (H342) is excited by ultraviolet light (UV), and emits blue fluorescence. Using H342 in place of SYBR-14 would overcome this limitation. Based on quantification of DNA content using H342, flow cytometric sperm sorting is the only effective means of sex preselection [14]. Furthermore, dead cells can be identified by adding PI to quench the H342 fluorescence of dead sperm so that they can be excluded from the sorting process by dead cell gating [2, 9, 14]. Because PI can also be excited by UV laser [2], modification of this method used for sperm sorting might be useful for evaluating sperm viability with a single UV excitation and two fluorescence detectors collecting H342 and PI fluorescence, respectively. To our knowledge, the H342=PI dual staining technique has been used only once to assess boar sperm viability [3]. Nevertheless, this technique has not been validated, and similar studies in other species have not been published. The rhesus monkey (Macaca mulatta), one species of nonhuman primate, has been extensively used in biomedical research for long history. Providing an accurate method for evaluating sperm functional capacity would be valuable for the study of sperm biology and the spermatozoa cryopreservation in this species. The objective of this study was to determine if H342=PI dual staining can be used to assess sperm viability in rhesus monkey semen excited by a single UV laser, and to extend this dual staining technique with a third stain, PNA-FITC, to simultaneously evaluate sperm viability and acrosome integrity of spermatozoa frozen using two different extenders commonly used for the monkey s spermatozoa cryopreservation [15, 17].

MATERIALS AND METHODS Materials Single UV Excitation of Hoechst 33342 and Propidium Iodide 373 SYBR-14 in LIVE=DEAD Sperm Viability Kit as component A (L-7011), and PI (P-1304) were purchased from Molecular Probes, Inc. (Eugene, OR, USA). All other reagents, including H342 (B-2261) and FITC-PNA (L-7381), were purchased from Sigma Chemical Co. (St. Louis, MO, USA). H342, PI, and FITC-PNA stock solutions were prepared 1 mg=ml in water; SYBR-14 stock solution prepared from the viability kit was diluted 1:50 with dimethyl sulfoxide before use. Semen Preparation A total of 10 semen samples were collected by penile electro-ejaculation from five rhesus monkeys, aged 8 13 years. Semen was collected into a disposable plastic test tube containing 2 ml of prewarmed TALP-Hepes [1]. The diluted semen was kept at 37 C water bath for 30 min to allow the clot to liquefy. After liquefaction, a small sample was taken out to examine sperm motility, and only samples with more than 75% motile spermatozoa were used. The qualifying semen was transferred into a 15-mL disposable plastic tube and washed twice with TALP-Hepes by centrifugation at 200 g for 10 min. Then the supernatant was removed and the sperm pellet was dispersed and mixed with a Pasteur pipette. Experiments 1a, 1b, and 1c: Validation of the H342=PI Dual Staining To provide quantifiable proportions of live and dead spermatozoa, the sperm pellet was resuspended in TALP-Hepes and divided into two equal parts. One part was maintained at 37 C, while the cells in the other part were killed by 2 cycles of freezing in liquid nitrogen for 5 min without cryoprotectant and thawing at 37 C. To obtain defined proportions of living and dead sperm, samples were made by combining aliquots of living and killed sperm cells at ratios of 100:0, 75:25, 50:50, 25:75, and 0:100. The mixtures were further diluted with TALP-Hepes and the final concentration was adjusted to approximately 5 10 6 =ml. Subsamples of 1 ml aliquots of the diluted semen were then transferred to 1.5-mL Eppendorf centrifuge tubes for fluorescent staining of each experiment as described below.

374 K. Cai et al. Experiment 1a: H342=PI Dual Staining for Flow Cytometric Analysis To assess sperm viability using flow cytometry, 6 ml volume of H342 and 8 ml volume of PI were added to each semen aliquot. Each sample was mixed by inverting three times and incubated for 15 min in a 37 C water bath shielded from light. Experiment 1b: H342=PI Dual Staining for Fluorescence Microscopic Analysis The staining procedure was identical to that described above except that the volume of H342 was reduced to 2 ml. After incubation, each sample was analyzed using fluorescence microscopy, at the same time of flow cytometric analysis. Experiment 1c: SYBR-14=H342=PI Triple Staining To further confirm the validity of H342=PI dual staining technique, 5 ml volume SYBR-14, 6 ml volume of H342 and 8 ml volume of PI were added to each 1 ml semen aliquot. Each sample was mixed by inverting three times and incubated for 15 min in a 37 C water bath shielded from light. After incubation, the samples were analyzed by flow cytometry. The accuracy of the H342=PI dual staining technique was validated by comparing H342 staining with SYBR-14 staining in these triple staining samples in the same flow cytometric analysis. Experiment 2: Evaluation of Cryopreserved Rhesus Monkey Sperm by H342=PI=FITC-PNA Triple Staining Each ejaculate was split into 2 aliquots and processed for cryopreservation using TTE and TEST extender, respectively, as described by Si et al. [15]. The thawed sample was washed twice and re-suspended with 1 ml of TALP-Hepes. To simultaneously estimate sperm viability and acrosomal status, 5 ml volume of FITC-PNA, 6 ml volume of H342 and 8 ml volume of PI were added to each semen aliquot. Each sample was gently mixed and incubated for 15 min in a 37 C water bath shielded from light, then analyzed by flow cytometry. Flow Cytometry Samples were analyzed on a Becton Dickinson FACS Vantage SE equipped with two Coherent Enterprise lasers. A multiline ultraviolet (UV) laser tuned to emit at 300 mw as the primary beam was used to excite H342 and PI. A 488 nm laser tuned to emit at 300 mw as the secondary beam was used to excite SYBR-14 and FITC-PNA. The H342 fluorescence intensity was collected on a linear scale; all others were collected on a logarithmic scale. Bandpass and dichroic filters for each detector were set up as follows: 390 LP in FL-1 for H342 fluorescence (FL-1), 630=22 BP in FL-2 for PI, a 560 SP dichroic separating FL-1 and FL-2, 530=30 BP in FL-4 for

Single UV Excitation of Hoechst 33342 and Propidium Iodide 375 SYBR-14 or FITC-PNA. Ten thousand events were collected and analyzed using CellQuest Software (Becton Dickinson). Background fluorescence of cellular debris was excluded by setting a threshold level using H342 fluorescence (FL-1) as the trigger (Channel 200). All fluorescent signals were shown as dot plot diagrams. In order to point out the different cell populations, regions were selected on the PI versus H342 fluorescence dot plots; for the PI versus SYBR-14, and PI versus FITC-PNA fluorescence dot plots, subpopulation were divided by quadrants, and the frequency of each subpopulation was quantified. Microscopic Examination Fluorescent staining was evaluated using an epifluorescence microscope (Zeiss Axiovert 200) equipped with triple filter sets, among which the FITC and UV filter sets were used. An 8 ml aliquot of each sample was pipetted on a clean slide, covered with a 22 22-mm coverglass, and observed at 400 magnification. Two replicates were made and a minimum of 200 spermatozoa per sample were counted for experiment 1b just after the completion of incubation. In addition, the general staining patterns of all experiments were visually monitored by epifluorescence microscopy. Statistical Analysis Coefficients of correlation between the percentage of Hoechst 33342 stained sperm (H342 þ =PI ), analyzed by flow cytometry and fluorescence microscopy and expected values of each ratios, were made using linear regression analysis. A paired sample t-test was used for the comparison between H342 and SYBR-14 stained population, and used to determine differences between the two extenders for sperm cryopreservation. All statistical analyses were performed using the SPSS software (version 10.0 for Windows, SPSS Inc., Chicago, IL, USA). The level of significance was set at P<0.05. RESULTS Experiments 1a, 1b, and 1c: Validation of the H342=PI Dual Staining Experiment 1a: H342=PI Dual Staining for Flow Cytometric Analysis Because debris particles do not contain DNA and therefore will not be stained by H342, threshold was set on H342 fluorescence in order to acquire all DNA-positive populations. When the threshold was set at channel 0 (no threshold), the separation between the cells and debris was very clear in the PI versus H342 fluorescence dot plot (Figure 1). Two cell

376 K. Cai et al. FIGURE 1 Dot plots of PI versus H342 fluorescence from one flow cytometric analysis when the threshold was set using H342 fluorescence as the trigger at channel 0. Population R1 and R2 represent sperm cells; population in the lower left corner represents debris in the sample. This debris population was electronically eliminated from data acquisition by setting a threshold level using H342 fluorescence as the trigger at channel 200. populations (R1 and R2), are located in the main position, while the population in the lower left corner represents cellular debris in the sample (Figure 1). All debris could be eliminated from analysis by setting a threshold level at channel 200 in the following experiments. To assess sperm viability, H342=PI dual stained sample was immediately analyzed by flow cytometry after incubation. Viable sperm are defined as those staining with H324 while resisting the uptake of PI. By flow cytometric analysis, two major populations of sperm were clearly identified, along with a transitional dual-stained population in the H342=PI dot plot. The living sperm have high H342 fluorescence and low PI fluorescence, whereas the dead sperm show high PI and low H342 fluorescence. The transitional dual-stained population connected with the dead population. These signals were interpreted as moribund cells. The average percentages of live sperm were 88.6, 67.6, 46, 24 and 0 for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the living and killed mixtures. The correlation between live population detected by flow cytometry with the expected viability was r ¼ 0.992 (P<0.001) (Figure 2). Experiment 1b: H342=PI Dual Staining for Fluorescence Microscopic Analysis The same samples were evaluated by fluorescence microscopy. The motile spermatozoa typically exhibited bright blue fluorescence over the sperm head and a very less intense blue over the tail, while nonmotile sperm typically exhibited pinkish over the sperm head. However, some

Single UV Excitation of Hoechst 33342 and Propidium Iodide 377 FIGURE 2 Regression plot for correlations between the percentages of live sperm analyzed by flow cytometry and the expected percentages. nonmotile sperm also fluoresced blue. A small portion of nonmotile sperm was stained with both H342 and PI. That is, the anterior region of the sperm nucleus retained the blue stain while the posterior portion of the nucleus progressively took up the red PI. Sperm showing partial or complete pinkish fluorescence were considered nonviable, while sperm showing complete blue fluorescence were considered viable. The mean percentages of viable sperm assessed by fluorescence microscope, for ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 of the living and killed mixtures were 85.0, 66.7, 44.4, 25.7 and 0.2, respectively. The correlation between the viable population detected by fluorescence microscopy with the expected percentages of living sperm was r ¼ 0.963 (P<0.001) (Figure 3). Data obtained by microscopic analysis of each sample were also FIGURE 3 Regression plot for correlations between the percentages of live sperm analyzed by fluorescence microscopy and the expected percentages.

378 K. Cai et al. compared to those obtained by flow cytometry. Paired sample t-test showed that the correlation between those two methods was r ¼ 0.964 (P<0.001). Experiment 1c: SYBR-14=H342=PI Triple Staining To further confirm the validity of H342=PI dual staining technique, the SYBR-14=H342=PI triple stained samples were evaluated by flow cytometry and were also monitored by fluorescence microscopy. By flow cytometric analysis, two major populations of sperm were clearly identified, along with a transitional dual-stained population in both H342=PI and SYBR-14=PI plots (Figure 4). The 2 dot plots were compared to detect each subpopulation by multicolor gating, which revealed that each population matched in both plots. In all samples, equivalent percentages of viable sperm were measured by the two dot plots (r ¼ 1, P<0.001). Virtually all H342 positive cells (H342 þ =PI ) were positive for SYBR-14 (SYBR-14 þ =PI ). The overall difference means of live sperm percentage was only 0.0240% (P ¼ 0.239) between these two plots. Microscopic analysis revealed that all those live sperm showing blue fluorescence in the sperm head in the UV light scope also exhibited green fluorescence in the visible scope, and all the moribund or dead sperm were also consistently corresponded between both scopes. FIGURE 4 Dot plots representing H342=PI=SYBR-14 triple stained spermatozoa. In panel A, dot plot of PI versus H342 fluorescence is indicated, displaying two main populations, along with a transitional dual-stained population. The living sperm have high H342 fluorescence and low PI fluorescence, whereas the dead sperm show high PI and low H342 fluorescence. The transitional dual-stained population is connected with the dead population. These signals were interpreted as moribund cells. In panel B, dot plot of PI versus SYBR-14 fluorescence is indicated. The distribution of sperm was divided into four quadrants. The proportion of viable sperm in panel A (R1) and B (upper right quadrant), as identified by negative PI staining, corresponded to each other, and were virtually identical.

Single UV Excitation of Hoechst 33342 and Propidium Iodide 379 FIGURE 5 Dot plots of PI versus H342 fluorescence representing H342=PI=FITC-PNA triple stained frozen-thawed spermatozoa cryopreserved in TTE (panel A) and TEST (panel B) extenders, respectively. Quadrant is set to distinguish the following sperm populations: live sperm with damaged acrosome appear in upper left quadrant; dead sperm with damaged acrosome appear in upper right quadrant; live sperm with intact acrosome appear in lower left quadrant; and dead spermatozoa with intact acrosome appear in lower right quadrant. Experiment 3: FITC-PNA=H342=PI Triple Staining By adding FITC-PNA to the H342=PI dual staining, the sperm viability and acrosomal status were evaluated simultaneously by flow cytometry. H342 fluorescence intensity was used to set a threshold level to exclude debris. PI=FITC-PNA dot plots of frozen-thawed samples are shown in Figure 5, which represent sperm cryopreserved using TTE (Figure 5a) and TEST (Figure 5b) extenders, respectively. The following sperm populations were identified in PI=FITC-PNA plot: FITC-PNA þ =PI (live sperm with damaged acrosome), FITC-PNA þ =PI (dead spermatozoa with damaged acrosome), FITC-PNA =PI (live sperm with intact acrosome) and FITC-PNA =PI þ (dead spermatozoa with intact acrosome). The effects of two extenders on post-thaw membrane and acrosome integrity in rhesus sperm are shown in Table 1. The percentage of live sperm with intact acrosome frozen in TTE was significantly higher than those frozen in TEST. The percentage of dead spermatozoa with damaged TABLE 1 Membrane and Acrosome Integrity of Frozen-Thawed Sperm Cryopreserved using Two Different Extenders Extenders FITC-PNA þ =PI FITC-PNA þ =PI þ FITC-PNA =PI FITC-PNA =PI þ TTE 1.1 0.4 a 42 5.6 a 54 5.2 a 2.2 1.1 a TEST 1.1 0.3 a 69 6.6 b 27 6.0 b 2.1 0.7 a a,b Numbers within columns with different superscripts differ, P<0.05.

380 K. Cai et al. acrosome was significantly higher when the spermatozoa were frozen in TEST. However, the percentages of live sperm with damaged acrosome and dead spermatozoa with intact acrosome were not different between samples frozen in TTE and TEST extenders. DISCUSSION In this report, a relatively simple and effective method of viability assessment using H342=PI dual staining by single UV laser excitation has been demonstrated (of course, PI also can be excited by visible light). This technique relies on exclusion of PI from the viable sperm, whereas H342 stains all of the cells. The correlation between expected viability and living sperm stained only with H342 was found to be very strong (r ¼ 0.992 for flow cytometry and 0.963 for microscopy). These relationships firmly establish the utility of this staining combination in determining viability of rhesus semen samples. This viability concept is also supported by the good correlation (r ¼ 0.964) between the results obtained by flow cytometry and fluorescence microscopy. It is important to note that the H342=PI dual staining condition used for flow cytometry is not optimal for fluorescence microscopy because the H342 blue fluorescence is too strong compared with red PI. By reducing the H342 final concentration to 2 mg=ml, it is easier to differentiate between viable blue and nonviable pinkish sperm using fluorescence microscopy. Although excitation with visible light (488 nm) is preferable to UV, the only effective means of sex preselection is flow cytometric sperm sorting, which is based on measuring the relative difference in DNA content of X- and Y- chromosome bearing sperm using H342 [14]. Furthermore, incubating sperm with H342 at levels required for sperm sorting does not impair sperm viability or their fertility [18]. Dead or moribund cells can be identified by adding PI to quench the H342 fluorescence of spermatozoa that have damaged membranes so that they can be excluded from the sorting process by dead-cell gating [2, 9, 14]. The current study confirms previous reports showing that H342 fluorescence intensity is reduced by incorporation of PI into the dead sperm. Various flow cytometric methods have been developed to measure sperm viability, among which the SYBR-14=PI dual staining is the mostly accepted [4, 5, 10]. The validity of H342=PI dual staining technique was confirmed by comparing H342 staining directly with SYBR-14 staining of the SYBR-14=H342=PI triple stained samples. The results indicated that live= dead sperm distributions were completely concordant in both H342=PI and SYBR-14=PI assays analyzed either by flow cytometry or monitored by fluorescence microscopy. These findings strongly indicate that this relatively inexpensive stain H342 could be used instead of SYBR-14 for

Single UV Excitation of Hoechst 33342 and Propidium Iodide 381 assessing sperm viability. Garner and colleagues [3] compared the staining efficacy of SYBR-14 and H342 in combination with PI for assessing epididymal and ejaculated boar sperm viability and found that the 2 staining combinations yielded similar results; however, there was less variation among sperm samples with the SYBR-14 staining than with the H342 staining. This was probably because, in that study, the H342=PI dual staining technique was not optimized. For example, higher concentration of cells (10 10 6 cells=ml) might not be stained sufficiently with lower concentrations of H342 dye (3.5 mg=ml), the H342 fluorescence was collected in log scale, and non-sperm events were gated out of analysis using light scatter parameters. While in the present study, higher concentration of H342 (6 mg=ml) was added to samples of lower concentration of sperm (5 10 6 cells=ml), which provide uniform staining for flow cytometric analyses without depressing viability. Furthermore, H342 fluorescence intensity was collected on a linear scale, and was used to set a threshold level to exclude debris. Because debris particles do not contain DNA and therefore will not be stained by H342, the non-stained debris do not trigger the cytometer, creating no signals to be processed. Generally, non-sperm events were gated out of analyses as judged on light scatter parameters; however, some debris particles present in the semen sample may have light scatter properties similar to those of the sperm. Furthermore, the flat, aspherical shape of mammalian sperm increases variability in light scatter [19]. Therefore, these non-sperm events cannot be completely excluded from analysis by light scatter gating. For this reason, the most effective method is to use dual staining protocol that stains live and dead cells simultaneously since fluorescent events can be safely regarded as sperm cells by the cytometer. For this purpose, esterase-based stain carboxyfluorescein diacetate, and intracellular ph indicator carboxy- SNARF-1 (SNARF) have been used in combination with PI to assess sperm viability [6, 11]. However, these probes have separate cellular targets compared to PI and fall short of distinguishing sperm from non-sperm particles. For the H342=PI dual staining technique, just like the SYBR-14=PI dual staining, staining time is not as critical, and these probes have the same cellular target, the sperm DNA, thus avoiding the ambiguity that arise from targeting different cellular organelles [4, 5, 10]. Although H342 require UV laser excitation not available in many of the flow cytometers, the spectral properties makes it very attractive to use in combination with other commonly used green probes excited at 488 nm, like PNA-FITC for multiparameter analysis. Spermatozoa must possess many attributes to fertilize an oocyte. Therefore, the only way to develop assays that can be consistently correlated with fertility is to develop unbiased assays that measure multiple sperm attributes simultaneously [8]. Evaluating viability and acrosome integrity of

382 K. Cai et al. spermatozoa simultaneously by flow cytometry may provide a valuable testing tool in predicting the fertility of semen sample. However, in the previous studies, non-stained debris particles will be incorrectly quantified as live acrosome intact sperm using the dual staining of PI and FITC-PSA or PI and FITC-PNA [10, 11]. In order to overcome this problem, Pe~na and colleagues [11] developed a new SNARF=PI=FITC-PSA triple staining method and Nagy and colleagues [10] extended the SYBR-14=PI dual staining with phycoerythrin conjugated PNA to evaluate viability and acrosome integrity simultaneously. In the present study, by adding FITC-PNA to the dual staining H342=PI, the sperm viability and the acrosome integrity were simultaneously evaluated and the debris was eliminated on the basis of H342 fluorescence intensity. Although the semen extender, TEST, is commonly and successfully used for cryopreservation of mammalian spermatozoa of different species, including cynomolgus monkey (Macaca fascicularis) [17], a previous study conducted in this laboratory showed that rhesus monkey spermatozoa suspended in TEST retained poor post-thaw motility (20% or less), while the post-thaw motility of spermatozoa frozen with TTE was more than 50% [15]. This study indicated that TTE offered better preservation of plasma and acrosome integrity than TEST and further confirmed that TTE is a reliable freezing extender for rhesus monkey sperm cryopreservation. In conclusion, the results demonstrate that the H342=PI dual staining technique, excited by a single UV laser, was effective in assessing rhesus monkey sperm viability and would be valuable for multiparameter flow cytometric analysis of sperm function. Unpublished observations indicated that this dual staining technique was also effective for evaluating sperm viability in mice, rat, bovine and cynomolgus monkey semen samples either by flow cytometry or fluorescence microscopy. Due to its ease, accuracy, and widespread applicability, the H342=PI technique has become the viability assay of choice in our laboratory. REFERENCES 1. Bavister BD, Leibfried ML, Lieberman G (1983): Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 28:235 247. 2. Boltz RC, Fischer PA, et al. (1994): Single UV excitation of Hoechst 33342 and ethidium bromide for simultaneous cell cycle analysis and viability determinations on in vitro cultures of murine B lymphocytes. Cytometry 15:28 34. 3. Garner DL, Dobrinsky JR, et al. (1996): Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14. Theriogenology 45:1103 1113. 4. Garner DL, Johnson LA, et al. (1994): Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J Androl 15:620 629. 5. Garner DL, Johnson LA (1995): Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 53:276 284. 6. Garner DL, Pinkel D, et al. (1986): Assessment of spermatozoa function using dual fluorescent staining and flow cytometric analyses. Biol Reprod 34:127 138.

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