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&#821...

<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<...

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&#39;,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&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-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&#39;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&#39;Aigle, France</td>
			<td>023862</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>Viability and concentration kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>024708</td>
			<td>kit for viability assessment</td>
		</tr>
		<tr>
			<td>Mitochondrial activity kit</td>
			<td>IMV Technologies, L&#39;Aigle, France</td>
			<td>024864</td>
			<td>kit for mitochondrial activity assessment</td>
		</tr>
		<tr>
			<td>Viability &#38; acrosome integrity kit</td>
			<td>IMV Technologies, L&#39;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>&#34;SION&#34;, 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.
<p clas...

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, ...

The goal of these procedures is to evaluate sperm membranes integrity using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analysis. These techniques might help answer key questions in the field of reproduction such as prediction of fertilization potential and quality of sperm sample. The main advantage of these techniques is that they enable precise evaluation of multiple sperm membranes which are known to associate with sperm fertilization potential.The implications of these techniques extend toward diagnosis of ejaculate quality as it enables more accurate evaluation of sperm plasma and acrosome membrane integrity as well as mitochondrial membrane potential. To begin this procedure, obtain approximately one to six milliliters of bull semen in a 15 milliliter tube at room temperature. Add six milliliters of NKM buffer pre-warmed to 37 degrees Celsius to each one milliliter of semen.Centrifuge at 600 times g for eight minutes. Repeat the centrifugation once or twice until the supernatant is clear. After this, immediately discard most of the supernatant, leaving approximately one centimeter of supernatant above the pellet.Carefully lean the tubes at a 30 degree angle in the incubator and wait 20 to 30 minutes to allow the spermatozoa to swim up. Using a micropipette, carefully remove the remaining one milliliter of supernatant which now contains the motile spermatozoa and transfer it to a fresh 1.5 milliliter tube. Keep the sperm at 37 degrees Celsius until ready to use.First, prepare all needed stock solutions as outlined in the text protocol. Next, transfer 133 microliters of the motile spermatozoa at a concentration of 25 million sperms per milliliter to a new 1.5 milliliter tube. Add 17 microliters of the DAPI working solution and incubate at 37 degrees Celsius for 10 minutes.Centrifuge at 1, 000 times g for five minutes. Discard the supernatant and add 100 microliters of NKM buffer to the pellet. Next, add 50 microliters of FITC-PSA, two microliters of JC-1, and three microliters of PI.Incubate at 37 degrees Celsius for 10 minutes.Centrifuge at 1, 000 times g for five minutes. Discard the supernatant and resuspend the pellet in 40 microliters of NKM buffer. Then transfer 10 microliters of the sample to a glass slide.Smear the sample and add a coverslip. After this, immediately begin visualizing the sample by epifluorescence microscopy with a 40X objective and a triple filter as outlined in the text protocol, using a digital camera to capture a separate image for each filter. Use the merge option in the camera software to merge the three images received from the filters, saving the new file in JPEG format.Open the merged image with the paint tool and use the brush option to mark the counted spermatozoa. Next, classify the spermatozoa based on the fluorescence emitted from each probe, making sure to evaluate at least 200 spermatozoa per slide. All cells should appear blue from the DAPI.Count the dead cells, which appear purple, to evaluate the viability. Then use the patterns of fluorescence staining to evaluate the acrosome status. Lastly, evaluate the mitochondrial membrane potential by distinguishing the spermatozoa with high mitochondrial membrane potential which exhibit a red-stained midpiece from the spermatozoa with low mitochondrial membrane potential which exhibit a green-stained midpiece.Count red and green midpieces separately and calculate their ratio. To begin the plasma membrane integrity evaluation, take the desired number of wells from the viability and concentration kit. Transfer the wells to the working base and cover them with a flexible lid to protect them from light.Add 199 microliters of buffered solution for cytometry to each well. Then add one microliter of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting. Cover the plate with the lid and incubate at 37 degrees Celsius for 10 minutes.After this, run the sample through the flow cytometer using the viability setting. To begin assessing the mitochondrial membrane potential, take out the desired number of wells from the mitochondrial activity kit. Transfer them to the working base.Add 10 microliters of absolute ethanol to each well and use a pipette to resuspend the powder present within the well. Add 190 microliters of PBS per well and homogenize by pipetting. Add 0.75 microliters of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting.Cover the plate with the lid and incubate at 37 degrees Celsius for 30 minutes. After this, run the sample through the flow cytometer using the mitochondrial activity setting. To begin assessing the acrosomal membrane integrity, take out the desired number of wells from the viability and acrosome integrity kit.Transfer the wells to the working base and cover them with a flexible lid to protect them from light. Add 200 microliters of buffered solution for cytometry to each well. Then add 0.7 microliters of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting.Cover the plate with the lid and incubate at 37 degrees Celsius for 45 minutes. After this, run the sample through the flow cytometer using the insight setting. In this study, semen quality is evaluated using different techniques, one of which evaluated sperm membranes using fluorometric probes.It is possible to evaluate the differences in sperm sample quality in terms of membrane integrity. For example, the ejaculate of bull number seven had a relatively low percentage of dead cells, a low proportion of sperm with pseudo-reacted acrosome, and higher mitochondrial membrane potential when compared to the ejaculate of bull number one. Samples are then evaluated for viability and mitochondrial activity.The histograms for these representative examples represent the ungated spermatozoa and debris and the gated spermatozoa. These histograms also represent the viable and dead cells and the distribution of spermatozoa to polarized and de-polarized mitochondrial membrane. The acrosome integrity is then evaluated with a ready-to-use kit and read with flow cytometry, dividing the area into three marker areas that represent negligible low fluorescing cells with intact unstained acrosome, low fluorescing cells with residual stained part of the acrosome, and highly fluorescing cells with disrupted acrosome.While attempting these procedures, it is important to make sure to prepare properly the initial sperm sample. These techniques can provide insight into sperm quality evaluation and can be applied to various organisms such as bovine, poultry, and human. Comparison between the two techniques, quadruple staining and flow cytometry indicated no significant differences for viability, mitochondrial membrane potential, and the acrosome integrity revealing that the two techniques produced matching results.

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

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Developmental Biology

12.9K visualizações.  Hebrew University of Jerusalem. O objetivo desses procedimentos é avaliar a integridade das membranas espermatozoides utilizando sondas fluorescentes específicas combinadas com microscopia de fluorescência ou análise de citometria de fluxo. Essas técnicas podem ajudar a responder a perguntas-chave no campo da reprodução, como a previsão do potencial de fertilização e a qualidade da amostra de espermatozoides. A principal vantagem dessas técnicas é que elas permitem uma avaliação precisa de múltiplas membranas...