The authors would like to thank &#34;SION&#34; Israeli company for artificial insemination and breeding (Hafetz-Haim, Israel) for their help and cooperation, and Ms. Li Na (IMV Technologies, L&#39;Aigle, France) for assistance with the instrument setup and training.

All of the experiments were performed in accordance with the 1994 Israeli guidelines for animal welfare. Bovine sperm was supplied by commercial Israeli company for artificial insemination and breeding. Ejaculates of 11 bulls were evaluated in this study.
1. Sperm Sample Preparation
NOTE: The procedure is based on the Roth laboratory&#39;s protocol1,3.
<ol>
	<li>Obtain approximately 1&#8211;6 mL of bull semen in a 15 mL tube at room temperature.</li>
	<li>To each 1 mL of semen, add 6 mL of prewarmed (at 37 &#176;C) NKM buffer (110 mM NaCl, 5 mM KCl, 20 mM MOPS [3-N-morphilino propanesulfonic acid; pH 7.4]) and centrifuge for 8 min at 600 x g, 1&#8211;2 times until the supernatant is clear. 
	NOTE: If sperm concentration or the initial volume are very high, split into two tubes at the first wash.</li>
	<li>Immediately remove and discard the clear supernatant and leave approximately 1 cm of the supernatant above the pellet.</li>
	<li>Carefully lean the tubes at a 30&#176; angle to increase the surface area for sperm to swim up and wait 20&#8211;30 min to allow spermatozoa to swim up at 37 &#176;C. 
	NOTE: Turbidity can be seen.</li>
	<li>Using a micropipette carefully, remove the upper 1 mL of the supernatant containing the motile spermatozoa to a new 1.5 mL tube.</li>
	<li>Keep the sperm at 37 &#176;C until use.</li>
	<li>Estimate the sperm count using a Neubauer hemocytometer. 
	NOTE: A different counting chamber can be used instead, but the counting is different.
	<ol>
		<li>To prevent spermatozoon movement, dilute 100 &#181;L of the motile spermatozoa with 10 mL of double distilled water (DDW) (1:100 dilution) in a 15 mL tube and mix gently.</li>
		<li>Load 10 &#181;L of the sample into each side of the hemocytometer and coverslip. Make sure to avoid bubble formation inside the chamber as this may result in an inaccurate sperm count.</li>
		<li>Observe under a compound microscope with a 20X objective. 
		NOTE: The full grid on a hemocytometer contains 9 large squares, each 1 mm2, and the coverglass rests 0.1 mm above the floor of the chamber. Thus, the volume over the central counting area is 0.1 mm3 or 0.1 &#181;L. The central area of the hemocytometer contains 25 medium squares and each medium square has 16 smaller squares with single lines.</li>
		<li>Count the total number of cells found in 4 medium corner squares and the central square. For higher precision, count two chambers (both sides of the Neubauer hemocytometer) and use the average to calculate cell concentration.</li>
		<li>Calculate the sperm count by multiplying the mean number obtained by 5 (to obtain the number of cells per counting area) and by 10,000 (to obtain the number of cells per 1 mL of diluted sample). Then multiply the obtained count by the dilution factor (1:100). 
		NOTE: For example, an average number of sperm counted in 5 of the 25 medium squares within the central counting area of two chambers is 150 ([152+148]/2). Thus, the mean number of sperms per chamber (or per 0.1 &#181;L) is 150 x 5 = 750. Multiply 750 by 10,000 to obtain the number of cells per 1 mL of diluted sample (7,500,000) and then multiply by 100 (dilution factor) to obtain 75 x 107 cells per mL of original semen sample.</li>
	</ol>
	</li>
</ol>
2. Technique #1: Simultaneous Assessment of Sperm Membranes Using Multiple Fluorescent Probes
NOTE: Sperm membranes (plasma, acrosomal and mitochondrial) were assessed as previously described by Celeghini et al.10, with some modifications. Epifluorescent microscopy was used, combined with a digital camera with excitation at 450-490 nm and emission at 515-565 nm using a triple filter.
<ol>
	<li>Prepare stock solutions.
	<ol>
		<li>Prepare 0.1 mg/mL DAPI stock solution by dissolving 5 mg of DAPI in 50 mL of phosphate buffered saline (PBS). Prepare 50 &#181;L aliquots and store at -20 &#176;C. Before use, dilute the stock solution with PBS at 1:10 (working solution; 10 &#181;g/mL).</li>
		<li>Prepare 1 mg/mL FITC&#8211;PSA stock solution by dissolving 1 mg of FITC&#8211;PSA in 1 mL of PBS. Prepare 50 &#181;L aliquots and store at -20 &#176;C. Before use, dilute the stock solution with PBS at 1:10 (working solution; 100 &#181;g/mL).</li>
		<li>Prepare 1 mg/mL JC-1 stock solution by dissolving 1 mg of JC-1 in 1 mL of dimethyl sulfoxide (DMSO). Prepare 10 &#181;L aliquots and store at -20 &#176;C. Before use, dilute the stock solution with DMSO at 1:10 (working solution; 0.1 mg/mL).</li>
		<li>Prepare the PI stock solution by dissolving 10 mg of PI in 400 &#181;L of PBS (giving 2.5 mg/mL). Store at +4 &#176;C. Dilute stock 1 with PBS at 1:20 (working solution; 0.125 mg/mL). Store at +4 &#176;C as a stock solution. 
		Caution: PI is a potential mutagen and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations.</li>
	</ol>
	</li>
	<li>Transfer 133 &#181;L of the motile spermatozoa (step 1.5) to a new 1.5 mL tube (25 x 106 sperm/mL). 
	NOTE: If the sample concentration is higher, dilute it in NKM buffer to achieve the required concentration; if the sample concentration of the swim up sample is lower, centrifuge the obtained supernatant after swimming up at 1,000 x g for 5 min, remove 0.5 mL of the supernatant and count the sperm again.</li>
	<li>Add 17 &#181;L of DAPI (working solution) and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge at 1,000 x g for 5 min and discard the supernatant.</li>
	<li>To the pellet, add 100 &#181;L of NKM buffer.</li>
	<li>Add 50 &#181;L of FITC&#8211;PSA, 2 &#181;L of JC-1 and 3 &#181;L of PI (working solutions) and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge at 1,000 x g for 5 min and remove the supernatant.</li>
	<li>To the pellet, add 40 &#181;L of NKM buffer and resuspend by pipetting.</li>
	<li>Transfer 10 &#181;L of the sample to a glass slide, smear and coverslip.</li>
	<li>Visualize immediately by epifluorescence microscopy (use 40x objective) with a triple filter, equipped with a digital camera and capture an image separately for each filter. 
	NOTE: There is no significance to the order of filters visualized.
	<ol>
		<li>Visualize under DAPI channel with excitation at 358 nm and emission at 461 nm.</li>
		<li>Visualize under FITC channel for green monomers with excitation at 450&#8211;490 nm and emission at 515&#8211;565 nm.</li>
		<li>Visualize under PI channel for red aggregates with excitation at 488 nm and emission at 590 nm.</li>
		<li>Visualize under JC-1 red aggregates with excitation at 559 nm, and emission in the range of 574&#8211;627 nm; JC-1 green monomers with excitation at 488 nm and emission in the range of 500&#8211;535 nm.</li>
	</ol>
	</li>
	<li>Merge the three images received from the filters in JPG/JPEG format, using the &#34;merge&#34; option of the camera software.</li>
	<li>Open the merged image with &#34;Paint&#34; tool and use the brush option to mark counted spermatozoa.</li>
	<li>Classify spermatozoa based on the fluorescence emitted from each probe:
	<ol>
		<li>In general evaluate at least 200 spermatozoa per slide&#8212;all cells appear blue (DAPI).</li>
		<li>Evaluate the viability by counting dead cells, which appear purple (PI [red] + DAPI [blue]) and calculate the percentage of dead cells (dead cells/total counted cells x 100).</li>
		<li>Evaluate acrosome status using the patterns of fluorescent staining (FITC&#8211;PSA). Calculate the percentages of the different patterns (intact, damaged or reacted acrosome cells/total counted cells x 100). 
		NOTE: Damaged acrosomal membrane appears as a fully stained, green acrosome cap; reacted acrosomal membrane shows residual green equatorial or upper staining; cells containing intact acrosomal membrane will not exhibit any green staining of the acrosomal region.</li>
		<li>Evaluate &#916;&#936;m by distinguishing spermatozoa with high &#916;&#936;m, which exhibit a red-stained midpiece, and spermatozoa with low &#916;&#936;m which exhibit a green-stained midpiece. Count red and green midpieces separately and calculate their ratio (red/green).</li>
	</ol>
	</li>
</ol>
3. Technique #2: Assessment of Sperm Membranes with Ready-To-Use Kits and Flow Cytometry
NOTE: Assessment of plasma membrane integrity, mitochondrial membrane potential and acrosomal membrane integrity was performed with ready-to-use flow cytometry kits containing lyophilized fluorochromes in each well. The procedure was performed according to the manufacturers with some modifications.
<ol>
	<li>Plasma membrane integrity evaluation
	<ol>
		<li>Take the desired number of wells from the package of viability and concentration kit (PI and SYbr14), transfer them to the working base and cover with a flexible lid (protect from light).</li>
		<li>Add 199 &#181;L of buffered solution for cytometry per well.</li>
		<li>Add 1 &#181;L of homogeneous semen at 57 x 106/mL (57,000 cells per well) and homogenize by pipetting.</li>
		<li>Cover the plate with the black lid.</li>
		<li>Incubate for 10 min at 37 &#176;C protected from light.</li>
		<li>Run the sample through the flow cytometer with the setting &#8216;viability&#8217;.</li>
	</ol>
	</li>
	<li>Mitochondrial membrane potential
	<ol>
		<li>Take the desired number of wells from the package of mitochondrial activity kit (JC-1), transfer them to the working base and cover with a flexible lid (protect from light).</li>
		<li>Add 10 &#181;L of absolute ethanol per well and pipette to resuspend the powder present within the well.</li>
		<li>Add 190 &#181;L of PBS per well and homogenize by pipetting.</li>
		<li>Add 0.75 &#181;L of homogeneous semen at 57 x 106/mL (50,000 cells per well) and homogenize by pipetting.</li>
		<li>Cover the plate with the black lid.</li>
		<li>Incubate for 30 min at 37 &#176;C protected from light.</li>
		<li>Run the sample through the flow cytometer with the setting &#701;mitochondrial activity&#8217;.</li>
	</ol>
	</li>
	<li>Acrosomal membrane integrity 
	NOTE: FITC&#8211;PSA staining (see Technique #1) enables the evaluation of 3 acrosome categories (intact acrosome, reacted acrosome and damaged acrosome). Using the flow cytometer and viability &#38; acrosome integrity kit (PI and FITC&#8211;PNA), the spermatozoa are separated into these 3 categories.
	<ol>
		<li>Take the desired number of wells from the package of viability &#38; acrosome integrity kit, transfer them to the working base and cover with a flexible lid (protect from light).</li>
		<li>Add 200 &#181;L of buffered solution for cytometry per well.</li>
		<li>Add 0.7 &#181;L of homogeneous semen at 57 x 106/mL (40,000 cells per well) and homogenize by pipetting.</li>
		<li>Cover the plate with the black lid.</li>
		<li>Incubate for 45 min at 37 &#176;C protected from light.</li>
		<li>Run the sample through the flow cytometer with the setting &#701;InCyte&#8217;.</li>
		<li>Analyze the resultant histogram by gating three marker areas according to fluorescence intensity, representing negligible, low-fluorescing cells with intact, unstained acrosome (R1), low-fluorescing cells with residual stained part of the acrosome (R2) and highly fluorescing cells with disrupted acrosome (R3). 
		NOTE: Use the &#34;analyzing files acquired using other modules&#34; section in the instrument user guide in order to create the three regions (R1, R2, R3).</li>
	</ol>
	</li>
</ol>
4. Technique #3: Assessment of Sperm Membranes Using Fluorescent Probes and Flow Cytometry
NOTE: Use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index).
<ol>
	<li>Prepare 1x annexin V binding buffer from 20x stock solution (dilute 500 &#181;L of annexin V binding buffer 20x stock solution with 9.5 mL of sterile distilled water).</li>
	<li>Estimate the sperm count using a Neubauer hemocytometer as described in section 1.7.</li>
	<li>Wash 106 spermatozoa in 1 mL of 1x annexin V binding buffer and centrifuge at 300 x g for 10 min.</li>
	<li>Aspirate the supernatant completely.</li>
	<li>Resuspend the pellet in 100 &#181;L of 1x annexin V binding buffer.</li>
	<li>Add 10 &#181;L of annexin V conjugated to FITC.</li>
	<li>Mix well and incubate for 15 min in the dark at room temperature.</li>
	<li>Wash spermatozoa by adding 1 mL of 1x annexin V binding buffer per 106 cells and centrifuge at 300 x g for 10 min.</li>
	<li>Aspirate the supernatant completely.</li>
	<li>Resuspend the cell pellet in 500 &#181;L of 1x annexin V binding buffer per 106 total cells.</li>
	<li>Add 1 &#181;g/mL PI immediately prior to analysis with a flow cytometer.</li>
	<li>Run the sample through the flow cytometer set on &#701;InCyte&#8217;.</li>
</ol>

<ol>
	<li>Komsky-Elbaz, A., Roth, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+herbicide+atrazine+and+its+metabolite+DACT+on+bovine+sperm+quality.">Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality.</a> Reprod Toxicol. 67, 15-25 (2016).</li><li>G&#252;rler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. 86 (2), 562-571 (2016).</li><li>Komsky-Elbaz, A., Saktsier, M., Roth, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxin+B1+impairs+sperm+quality+and+fertilization+competence.">Aflatoxin B1 impairs sperm quality and fertilization competence.</a> Toxicology. 393, 42-50 (2018).</li><li>Beltr&#225;n, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ion+Channels+in+the+Sperm+Acrosome+Reaction.">Role of Ion Channels in the Sperm Acrosome Reaction.</a> Adv Anat Embryol Cell Biol. 220, 35-69 (2016).</li><li>Breitbart, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+sperm+capacitation+and+acrosome+reaction.">Signaling pathways in sperm capacitation and acrosome reaction.</a> Cell Mol Biol (Noisy-le-grand). 49 (3), 321-327 (2003).</li><li>Almadaly, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+factors+affecting+the+results+of+staining+frozen-thawed+fertile+and+subfertile+Japanese+Black+bull+spermatozoa+for+acrosomal+status.">Methodological factors affecting the results of staining frozen-thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status.</a> Anim Reprod Sci. 136 (1-2), 23-32 (2012).</li><li>Jankovicov&#225;, J., Simon, M., Antal&#237;kov&#225;, J., Horovsk&#225;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrosomal+and+viability+status+of+bovine+spermatozoa+evaluated+by+two+staining+methods.">Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods.</a> Vet Hung. 56 (1), 133-138 (2008).</li><li>Lybaert, P., Danguy, A., Leleux, F., Meuris, S., Lebrun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methodology+for+the+detection+and+quantification+of+the+acrosome+reaction+in+mouse+spermatozoa.">Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.</a> Histol Histopathol. 24 (8), 999-1007 (2009).</li><li>Whitfield, C. H., Parkinson, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+fertility+of+bovine+semen+and+in+vitro+induction+of+acrosome+reactions+by+heparin.">Relationship between fertility of bovine semen and in vitro induction of acrosome reactions by heparin.</a> Theriogenology. 38 (1), 11-20 (1992).</li><li>Celeghini, E. C. C., de Arruda, R. P., de Andrade, A. F. C., Nascimento, J., Raphael, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Techniques+for+Bovine+Sperm+Simultaneous+Fluorimetric+Assessment+of+Plasma,+Acrosomal+and+Mitochondrial+Membranes.">Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes.</a> Reprod Domest Anim. 42 (5), 479-488 (2007).</li><li>Ramalho-Santos, J., Varum, S., Amaral, S., Mota, P. C., Sousa, A. P., Amaral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functionality+in+reproduction:+from+gonads+and+gametes+to+embryos+and+embryonic+stem+cells.">Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.</a> Hum Reprod. 15 (5), 553-572 (2009).</li><li>Eddy, E. M., O&#8217;Brien, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The spermatozoon. , (1994).</li><li>Gallon, F., Marchetti, C., Jouy, N., Marchetti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functionality+of+mitochondria+differentiates+human+spermatozoa+with+high+and+low+fertilizing+capability.">The functionality of mitochondria differentiates human spermatozoa with high and low fertilizing capability.</a> Fertil Steril. 86 (5), 1526-1530 (2006).</li><li>Espinoza, J. a., Paasch, U., Villegas, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+potential+disruption+pattern+in+human+sperm.">Mitochondrial membrane potential disruption pattern in human sperm.</a> Hum Reprod. 24 (9), 2079-2085 (2009).</li><li>H&#252;ttemann, M., Lee, I., Pecinova, A., Pecina, P., Przyklenk, K., Doan, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+oxidative+phosphorylation,+the+mitochondrial+membrane+potential,+and+their+role+in+human+disease.">Regulation of oxidative phosphorylation, the mitochondrial membrane potential, and their role in human disease.</a> J Bioenerg Biomembr. 40 (5), 445-456 (2008).</li><li>Sellem, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+combinations+of+in+vitro+quality+assessments+to+predict+fertility+of+bovine+semen.">Use of combinations of in vitro quality assessments to predict fertility of bovine semen.</a> Theriogenology. 84 (9), 1447-1454 (2015).</li><li>Odhiambo, J. F., Sutovsky, M., DeJarnette, J. M., Marshall, C., Sutovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+ubiquitin-PNA+based+sperm+quality+assay+for+semen+evaluation+by+a+conventional+flow+cytometer+and+a+dedicated+platform+for+flow+cytometric+semen+analysis.">Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis.</a> Theriogenology. 76 (6), 1168-1176 (2011).</li><li>Barrier Battut, I., Kempfer, A., Becker, J., Lebailly, L., Camugli, S., Chevrier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+fertility+prediction+model+for+stallion+semen,+including+flow+cytometry.">Development of a new fertility prediction model for stallion semen, including flow cytometry.</a> Theriogenology. 86 (4), 1111-1131 (2016).</li></ol>

The authors declare that there are no conflicts of interest.

Sperm fertilization potential depends on multiple factors reflecting its quality. A high concentration of spermatozoa and a high proportion of highly progressively motile spermatozoa might be considered high-quality semen. Nevertheless, such an evaluation does not take into account other cellular and functional parameters. The use of &#39;bench-top&#39; microcapillary flow cytometer can be easily adapted to evaluation of various sperm structures using fluorescent probes, as previously shown by others17&#160;and demonstrated herein (Technique #3). For example, sperm acrosome integrity is highly important for the occurrence of successful natural fertilization and therefore, precise evaluation of acrosomal status is warranted. Such an evaluation can be easily performed by classification of acrosome status using the patterns of fluorescent staining (FITC-PSA, FITC-PNA, i.e., Technique #1, as previously described)1,3.&#160;In particular, it is highly important to determine the proportion of sperm with intact acrosome (i.e., exhibits an unstained acrosome) relative to those with damaged acrosome. With respect to the latter, sperm with damaged acrosome can exhibit (i) a fully stained acrosomal cap, which indicates that the membrane is damaged, enabling the dye to flow through the membrane into the acrosome vesicle; (ii) acrosome-reacted sperm that exhibit only residual acrosome content, indicating that the AR has already occurred (i.e., pseudo AR). It should be noted that such an evaluation can also be performed with the dedicated flow cytometer.
The ready-to-use viability &#38; acrosome integrity kit defines both sperm viability (viable or dead) and acrosomal integrity (intact or disrupted). Here, we suggest using the dedicated flow cytometer to define the three aforementioned acrosomal statuses (i.e., intact, damaged, reacted). We adapted the microcapillary flow cytometer platform for more accurate evaluation, which identifies the acrosome-reacted sperm (i.e., low fluorescence) while excluding them from those with disrupted acrosome (high fluorescence), rather than including them with those having an intact acrosome. This gives an accurate proportion of sperm with functional or nonfunctional acrosome. Sperm with reacted acrosome as well as disrupted acrosomal membrane have lost their ability to fertilize the oocyte. Furthermore, accurate analysis might shed light on the mechanism underlying acrosome alteration, i.e., damaged acrosome membrane vs. pseudo acrosome activation.
We compared the results obtained with Technique #1 and Technique #2, and found great compatibility between them, in particular in the evaluation of viability and &#916;&#936;m&#160;(Table 1). One of the main advantages of using the the dedicated flow cytometer is the large number of evaluated spermatozoa relative to the small number of spermatozoa that are evaluated in practice by fluorescence microscopy and probes (thousands vs. hundreds, respectively). Moreover, the latter procedure is time-consuming and subjective, even when performed by an experienced observer. As flow cytometry only detects particle-associated fluorescence, there is no need to wash the unbound probe from the solution, which is a time-consuming step17. On the other hand, the fluorimetric assessment of sperm membranes described in Technique #1 enables simultaneous assessment of multiple membranes. We were able to use as many as four fluorescent probes together1,3.
Finally, it should be noted that the dedicated flow cytometer was developed as an open assay module, providing all of the basic tools for sample acquisition and data analysis. The acquisition function enables collecting various types of information from a cell sample and therefore allows adaptation for more accurate evaluation, as shown here for acrosome status and apoptotic index.
In conclusion, the methodologies described in this paper are very useful for the evaluation of semen quality. Examining spermatozoon membranes is highly important for determining sperm fertilization competence.

Integrity and functionality of sperm membranes are a few of the factors indicating sperm viability and fertilization potential. The plasma membrane acts as a barrier between intracellular and extracellular compartments, thereby maintaining the cellular osmotic equilibrium1. Any stress that induces damage to the plasma membrane integrity might impair homeostasis, reduce viability and fertilization capacity, and increase cell death. For instance, cryopreservation reduces sperm viability due to damage to its plasma membrane, as a result of temperature changes and osmotic stress2. We previously reported that exposing bull sperm to low concentrations of foodborne contaminants such as the pesticide atrazine, its major metabolite diaminochlorotriazine or the mycotoxin aflatoxin B1, reduces sperm viability1,3. This was determined by labeling the double-stranded DNA with DAPI in combination with PI, which binds to the DNA of cells with a damaged plasma membrane.
Acrosome reaction (AR) involves fusion&#160;of the outer acrosome membrane and the overlying plasma membrane resulting in the release of acrosomal enzymes4,5. These are essential events for zona-pellucida penetration and further merging of the sperm with the oocyte6. Therefore, evaluation of acrosomal membrane integrity constitutes a useful parameter to evaluate the semen quality and male fertility7,8,9. Several fluorescent techniques are suitable for the verification of acrosome integrity, FITC-PNA or FITC-PSA8,10. In our previous studies, using the patterns of FITC-PSA staining1,3, we provided accurate definitions for (i) intact acrosome, (ii) damaged acrosome membrane and (iii) reacted acrosome. In the current report, we evaluate acrosome status using sperm-dedicated flow cytometry and compare the results to those using fluorescence microscopy.
The mitochondria are multifunctional organelles involved in, among other things, ATP synthesis, reactive oxygen species production, calcium signaling and apoptosis. Physiological dysfunctions, including male and female infertility, are associated with altered mitochondrial function11.Sperm mitochondria are arranged in the midpiece and play a crucial role in sperm motility12. It is well accepted that high mitochondrial membrane potential (&#916;&#936;m) is associated with normal motility and high fertilization capacity13. In contrast, low &#916;&#936;m&#160;is associated with an elevated level of reactive oxygen species and reduced fertilization rate14. Nonetheless, various environmental compounds, for instance endocrine disruptors, can induce cellular stress and lead to a transient increase in &#916;&#936;m, hyperpolarization1,3, increased production of free radicals and eventually, apoptosis15. The fluorescent probe 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) enables examining for example, the effects of foodborne toxins on sperm &#916;&#936;m1,3.
Standard spermiograms, based on physiological and morphological parameters, are not good enough to predict semen quality. More accurate methods are required to ensure sperm quality. Here, we provide two feasible methods to determine sperm quality based on evaluations of sperm membranes: simultaneous quadruple staining with specific fluorescent probes and fluorescence microscopy, described in our studies1,3 and advanced sperm-dedicated flow cytometry, recently utilized in our laboratory, and already being used by others16,17,18.

<table><tbody><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>MOPS [3-N-morphilino propanesulfonic acid]</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P5493</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2438</td><td></td></tr><tr><td>Ethanol absolute</td><td>Sigma</td><td>64-17-5</td><td></td></tr><tr><td>Hemacytometer</td><td>Neubauer Germany</td><td></td><td>hemocytometer</td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Sigma</td><td>D9542</td><td>fluorescent probe</td></tr><tr><td>PI (propidium iodide )</td><td>Sigma</td><td>P4170</td><td>fluorescent probe</td></tr><tr><td>FITC-PSA (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin )</td><td>Sigma</td><td>L0770</td><td>fluorescent probe</td></tr><tr><td>JC-1 (5,5&#039;,6,6&#039;-tetra-chloro-1,1&#039;,3,3&#039;-tetraethylbenzimidazolyl carbocyanine iodide)</td><td>ENZOBiochem, New York, NY, USA</td><td>ENZ52304</td><td>fluorescent probe</td></tr><tr><td>Annexin V conjugated to FITC</td><td>MACS, Miltenyi Biotec</td><td>130-093-060</td><td>fluorescent probe</td></tr><tr><td>Annexin V binding buffer 20x stock solution</td><td>MACS, Miltenyi Biotec</td><td>130-092-820</td><td>buffer</td></tr><tr><td>Nikon Eclipse, TE-2000-u</td><td>Nikon, Tokyo, Japan</td><td></td><td>inverted fluorescence microscope</td></tr><tr><td>Nis Elements</td><td>Nikon, Tokyo, Japan</td><td></td><td>software</td></tr><tr><td>Nikon DXM1200F</td><td>Nikon, Tokyo, Japan</td><td></td><td>digital camera</td></tr><tr><td>Guava EasyCyte Plus</td><td>IMV Technologies, L&#039;Aigle, France</td><td></td><td>microcapillary sperm flow cytometer</td></tr><tr><td>CytoSoft</td><td>Guava Technologies Inc., Hayward, CA, USA; distributed by IMV Technologies</td><td></td><td>software</td></tr><tr><td>Buffered solution for cytometry</td><td>IMV Technologies, L&#039;Aigle, France</td><td>023862</td><td>buffer</td></tr><tr><td>Viability and concentration kit</td><td>IMV Technologies, L&#039;Aigle, France</td><td>024708</td><td>kit for viability assessment</td></tr><tr><td>Mitochondrial activity kit</td><td>IMV Technologies, L&#039;Aigle, France</td><td>024864</td><td>kit for mitochondrial activity assessment</td></tr><tr><td>Viability &amp;amp; acrosome integrity kit</td><td>IMV Technologies, L&#039;Aigle, France</td><td>025293</td><td>kit for acrosome integrity assessment</td></tr><tr><td>JMP-13</td><td>SAS Institute Inc., 2004, ary, NC, USA</td><td></td><td>software</td></tr><tr><td>Bovine sperm</td><td>&quot;SION&quot;, Israeli company for artificial insemination and dreeding, Hafetz-Haim, Israel</td><td></td><td></td></tr></tbody></table>

fluorimetric techniques for the assessment of sperm membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Figure 1 shows simultaneous fluorimetric assessment of sperm membranes (plasma, acrosomal and mitochondrial) using PI, DAPI, FITC-PSA and JC-1. Assessment of sperm membranes using simultaneous staining with four fluorescent probes allows, for example, evaluating the proportion of sperm in each category&#8212;live vs. dead; high vs. low &#916;&#936;m; intact vs. damaged acrosome&#8212;simultaneously for each spermatozoon.
Figure 2 presents results of sperm membrane evaluation using fluorimetric probes. Only semen that contained at least 80% motile spermatozoa were used in the experiment. At least 200 cells were examined per bull. It was possible to evaluate the differences in sperm sample quality in terms of membrane integrity. For example, the ejaculate of bull no. 7 had a relatively low percentage of dead cells, a low proportion of sperm with pseudo reacted acrosome and higher mitochondrial membrane potential, as compared to the ejaculate of bull no. 1.
Figure 3 shows representative samples evaluated for viability (Figure 3A-3C) and mitochondrial activity (Figure 3D-3F). Fluorescence intensities of the samples were evaluated by a dedicated microcapillary sperm flow cytometer, with dedicated software. This flow cytometer contains one solid-phase blue laser (448 nm) and two photodiodes: forward scatter and side scatter. It specifically measures sperm emission properties with three photomultiplier tubes (green: 525/30 nm, yellow: 583/26 nm; red: 655/50 nm) and accommodates optical filters and splitters16. It enables evaluation of 5,000 spermatozoa per analysis.
The viability evaluation kit contains a probe with differential permeability to viable (intact plasma membrane) and dead (damaged plasma membrane) spermatozoa (Figure 3C). Sperm &#916;&#936;m&#160;was assessed using a kit that distinguishes between polarized mitochondrial membrane (fluorescence appearing in orange) and depolarized mitochondrial membrane (fluorescence appearing in green) (Figure 3F).
Figure 4 presents an evaluation of acrosome integrity performed with the ready-to-use kit, read with the flow cytometry (Figures 4A-4C), dividing the resultant histogram of gated spermatozoa into three marker areas, representing negligible low-fluorescing cells with intact, unstained acrosome (R1), low-fluorescing cells with residual stained part of the acrosome (R2), and highly fluorescing cells with disrupted acrosome (R3).
Table 1 presents a comparison of the two fluorimetric techniques for assessment of sperm membranes. The same sperm samples from three different bulls were evaluated for viability, mitochondrial membrane potential (&#916;&#936;m) and acrosome integrity using simultaneous quadruple staining as well as flow cytometry. This comparison is highly important, as it shows the matching results using each of the two techniques. Data were analyzed by an analysis and Student&#39;s t-test. No statistically significant differences were observed.
Figure 5 shows a representative sample evaluated for apoptosis using annexin V (AV) and propidium iodide (PI) fluorochromes. Use of these two probes enables distinguishing among four patterns indicating viable cells (AV-, PI-), early apoptotic cells (AV+, PI-), apoptotic cells (AV+, PI+) and necrotic cells (AV-, PI+).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58622/58622fig1.jpg" /> 
Figure 1: Epifluorescence photomicrography of spermatozoa stained simultaneously with several fluorescent probes. (A) Simultaneous staining with four probes PI, DAPI, FITC-PSA and JC-1) (B) Live spermatozoon with DAPI staining of nucleus and high mitochondrial membrane potential (&#916;&#936;m), stained with JC-1 probe. (C) Dead spermatozoon with damaged plasma membrane stained with PI probe, damaged acrosome stained with FITC-PSA probe and low &#916;&#936;m. (D) Live, acrosome-reacted spermatozoon with residual equatorial staining and low &#916;&#936;m. (E) Live, acrosome-reacted spermatozoon with residual upper staining and high &#916;&#936;m. Scale bars = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58622/58622fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58622/58622fig2.jpg" /> 
Figure 2: Evaluation of bull sperm membranes using fluorimetric probes.&#160;(A)&#160;Sperm viability was determined with fluorescent probes 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). (B)&#160;Acrosome status was determined according to FITC-PSA staining patterns. Presented are the proportion of spermatozoa with reacted acrosome. (C)&#160;Mitochondrial membrane potential (&#916;&#936;m) was evaluated using with JC-1 fluorescent probe and presented as the ratio between mean proportion of red-stained (high potential) and green-stained (low potential) sperm. Data are presented as percent of cells out of total evaluated cells. At least 200 spermatozoa were analyzed per bull. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58622/58622fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58622/58622fig3.jpg" /> 
Figure 3: Viability (A-C) and mitochondrial activity (D-F) fluorescence assessment of representative samples measured by EasyCyte flow cytometer. Histograms represent ungated spermatozoa and debris (A, D), gated spermatozoa (B, E), distribution of spermatozoa to viable (green) and dead (red) cells (C), and distribution of spermatozoa to polarized (yellow) and depolarized (green) mitochondrial membrane (F). Scale bars = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58622/58622fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58622/58622fig4.jpg" /> 
Figure 4: Fluorescence assessment of acrosome integrity of representative samples measured by EasyCyte flow cytometer.&#160;(A)&#160;Histogram of ungated spermatozoa and debris. (B, C) Histograms of gated spermatozoa with evaluation of acrosome integrity performed with ready-to-use kit, read with adapted setting &#39;InCyte&#39;, dividing the resultant histogram of gated spermatozoa into three marker areas, representing negligible, low-fluorescing cells with intact, unstained acrosome (R1), low-fluorescing cells with residual stained part of the acrosome (R2) and highly fluorescing cells with disrupted acrosome (R3).&#160;Scale bars = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58622/58622fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Average No of Cells</td>
			<td colspan="2">Vialbility</td>
			<td colspan="3">Mitochondrial Membrane potential</td>
			<td colspan="3">Acrosome Integrity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Viable</td>
			<td>Dead</td>
			<td>Depolarized</td>
			<td>Polarized</td>
			<td>Red/Green Ratio</td>
			<td>Intact Acrosome</td>
			<td>Reacted Acrosome</td>
			<td>Disrupted Acrosome</td>
		</tr>
		<tr>
			<td>Quadruple Staining</td>
			<td>253</td>
			<td>32.7 &#177; 1.53%</td>
			<td>67.3 &#177; 1.53%</td>
			<td>65.7 &#177; 2.25%</td>
			<td>34.3 &#177; 2.52%</td>
			<td>0.5 &#177; 0.06</td>
			<td>37.3 &#177; 7.2%</td>
			<td>38.0 &#177; 5.7%</td>
			<td>24.3 &#177; 3.0%</td>
		</tr>
		<tr>
			<td>Flow Cytomtery</td>
			<td>5,000</td>
			<td>32.3 &#177; 2.08%</td>
			<td>67.7 &#177; 2.08%</td>
			<td>65.0 &#177; 1.00%</td>
			<td>35.0 &#177; 1.00%</td>
			<td>0.5 &#177; 0.02</td>
			<td>39.5 &#177; 5.7%</td>
			<td>39.5 &#177; 6.5%</td>
			<td>21.0 &#177; 8.0%</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of the two fluorimetric techniques for assessment of sperm membranes. The same sperm samples were evaluated for viability, mitochondrial membrane potential and acrosome integrity using simultaneous quadruple staining and flow cytometry. Data are presented as mean proportion &#177; SD of the examined cells, calculated for 3 replicates.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58622/58622fig5.jpg" /> 
Figure 5: Annexin V and PI fluorescence of a representative sample measured by a flow cytometer. Histograms represent (A)&#160;ungated spermatozoa and debris and (B)&#160;distribution of the gated spermatozoa to early apoptotic (AV+, PI-), apoptotic (AV+, PI+), viable (AV-, PI-) and necrotic (AV-, PI+) cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58622/58622fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fluorimetric Techniques for the Assessment of Sperm Membranes at JoVE.com

Fluorimetric Techniques for the Assessment of Sperm Membranes

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

fluorimetric-techniques-for-the-assessment-of-sperm-membranes

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.2K Views.  Hebrew University of Jerusalem. Het doel van deze procedures is om de integriteit van spermamembranen te evalueren met behulp van specifieke fluorescerende sondes in combinatie met fluorescentiemicroscopie of flow cytometrieanalyse. Deze technieken kunnen helpen bij het beantwoorden van belangrijke vragen op het gebied van reproductie, zoals voorspelling van bevruchting potentieel en de kwaliteit van sperma monster. Het belangrijkste voordeel van deze technieken is dat ze nauwkeurige evaluatie van meerdere spermamembranen d...

Video: Fluorimetric Techniques for the Assessment of Sperm Membranes

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Biology

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...

Medicine

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for ...

Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization

Source: Bird, L. E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52357">Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli</a>. J. Vis. Exp. (2015)
In this video, we demonstrate a fluorescence-detection size-exclusion chromatography technique to screen for green fluorescent protein (GFP)-fused membrane protein stability in Escherichia coli following detergent solubilization. Proteins exhibiting a symmetrical peak in the size-exclusion profile with little or no free GFP indicate purified proteins with minimum degradation and aggregation, which can subsequently be selected for structure-function analysis.

Source: Bird, L. E., et al. Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli. J. Vis. Exp. (2015)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

An In Vitro Fluorescence-Based Assay to Measure Plasma Membrane Resealing Efficiency

To set up a high throughput fluorescence-based assay for the repair permissive conditions, wash the cells two times with 200 microliters of 37 degrees Celsius M1 medium per well, and add 100 microliters of fresh 37 degrees Celsius M1 medium supplemented with 30-micromolar propidium iodide after discarding the second wash.
For repair restrictive conditions, wash the cells one time with 200 microliters of 37 degrees Celsius M2 medium supplemented with 5-millimolar EGTA per well, followed by one wash with 200 microliters of M2 medium alone.
After discarding the second wash, add 100 microliters of fresh 37 degrees Celsius M2 medium supplemented with 30-micromolar propidium iodide per well. Then, image the plate under transmitted light, GFP, and PI, as just demonstrated. While the plate is being imaged, place a 96-well round-bottom polypropylene microplate on ice, and configure the plate as just demonstrated for the previous plate.
For repair permissive conditions, add 100 microliters of ice-cold M1 medium supplemented with 60 micromolar propidium iodide per well, followed by the addition of 100 microliters of ice-cold M1 with or without 4x listeriolysin O per well.
For repair restrictive conditions, add 100 microliters of ice-cold M2 medium supplemented with 60 micromolar propidium iodide per well, followed by the addition of 100 microliters of ice-cold M2 with or without 4x listeriolysin O per well.
It is imperative to prepare listeriolysin O at the appropriate experimental concentration on ice approximately 5 minutes prior to the start of the kinetic assay when plate 1 is on ice and plate 2 is being prepared.
When the first plate is finished imaging, immediately transfer the plate onto a sheet of aluminum foil on ice. After 5 minutes, transfer 100 microliters from each well of the second plate to the appropriate corresponding well in the first cooled plate, inserting the pipette tips below the meniscus of solution in each well before ejecting the volume without mixing for proper distribution of the toxin.
Allow the toxin to bind to the host cells for 1 minute, and immediately transfer the first plate to the plate reader for the postkinetic assay using the spectrofluorometer mode. At the end of the kinetic assay, immediately acquire a postkinetic imaging of the plate image as demonstrated for the prekinetic imaging.

Fluorimetric Techniques for the Assessment of Sperm Membranes

Samenvatting

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