









































Type of the Paper (Article


 

  

 

 
Cluj Vet J 2023 vol 28, issue 2 http://clujveterinaryjournal.ro 

Review 

Flow cytometry and its use in modern human and veterinary 
andrology 
Diana Cenariu 1,*, Jon Thor Bergthorsson2, Victor Greiff3, BogdanȚigu1, Valentin Toma1 and Mihai Cenariu 4 

1 Research Center for Advanced Medicine – MedFuture, Iuliu Hatieganu University of Medicine and Phar-
macy Cluj-Napoca, Romania; diacenariu@gmail.com 

2 University of Iceland, Reykjavik, Iceland; jon.bergthorsson@gmail.com  
3 University of Oslo, Norway; victor.greiff@medisin.uio.no 
4 University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania; mcenariu@yahoo.es 
* Correspondence: diacenariu@gmail.com; 

Abstract: Fertility of a male is sometimes difficult to assess and results obtained using regular sperm analysis methods are often 
inconclusive. Flow cytometry was proven to generate crucial information, that allows specialists to conclude upon the reproductive 
capacity of an individual. Together with computer assisted sperm analysis CASA systems, flow cytometers allow an in-depth anal-
ysis of fresh, chilled or frozen/thawed semen, which is currently essential for research purposes, but also for diagnosis of various 
male-related fertility disorders, both in veterinary and human medicine. Several parameters can be assessed using this method, such 
as sperm viability, acrosome integrity, mitochondrial activity, concentration and total sperm number, DNA fragmentation, capacita-
tion, oxidative stress, etc. This paper provides a rapid reference to specialists involved in semen analysis by flow cytometry, regarding 
semen sample preparation, instrument setup, and evaluation of the most important sperm parameters (viability, acrosome reaction 
and mitochondrial activity). 

Keywords: flow cytometry; mammalian sperm; viability; acrosome integrity; mitochondrial activity. 

 

1. Introduction 
Flow cytometers are made up of several independent systems that are interconnected 

to yield the final results, such as fluidics, that allows a single-stranded alignment of 
events, optics, made up of several lasers, light filters and detectors as well as electronics, 
which allows conversion of optical signals into electronic information which can be stored 
and analysed by a computer, equipped with a dedicated software. As such, flow cytome-
try represents a powerful technique that allows a complex and prompt evaluation, or even 
separation, of single cells (called events) found in suspension, making it therefore very 
suitable for sperm analysis. Cells can be thus evaluated regarding their size (forward scat-
ter), internal complexity usually given by their granularity (side scatter) as well as fluo-
rescent intensity (when stained with fluorescent antibodies or dyes that bind to the nu-
cleus, cytoplasm, or membrane) [1]. Since fertility of a male is sometimes difficult to assess 
and results obtained using regular sperm analysis methods are often inconclusive, flow 
cytometry was proven to generate crucial information, that allows specialists to conclude 
upon the reproductive capacity of an individual. Several parameters can be assessed us-
ing this method, such as sperm viability, acrosome integrity, mitochondrial activity, con-
centration and total sperm number, DNA fragmentation, capacitation, oxidative stress, 
etc. [2]. This paper is aimed at providing a rapid reference to specialists involved in semen 
analysis by flow cytometry, regarding semen sample preparation, instrument setup, and 
evaluation of the most important sperm parameters (viability, acrosome reaction and mi-
tochondrial activity).  

 
 
 

Received: 03.09.2023 

Accepted: 17.10.2023 

Published: 20.10.2023 

DOI: 10.52331/cvj.v28i2.49 

 

 

 

Copyright: © 2023 by the authors. 

Submitted for possible open access 

publication under the terms and 

conditions of the Creative Commons 

Attribution (CC BY) license 

(http://creativecommons.org/licenses

/by/4.0/). 



Cluj Vet J 2023, vol 28, issue 45  
 

2. Semen Sample Preparation 
Semen analyzed by flow cytometry may originate from fresh ejaculates, collected by any of the com-

monly used methods (masturbation, artificial vagina, electroejaculation, etc.) but may also be obtained after 
flushing of epididymis (from dead/slaughtered animals or following castration). Frozen semen samples may 
also be investigated by flow cytometry and such studies are of particular interest to assess efficacy of the 
freezing method or suitability of the extender used [3].  

One of the biggest issues that has to be taken into consideration is interference between components of 
the seminal plasma or extender and staining dyes. In cases where this represents a serious concern, the most 
suitable method of sample clean-up or washing has to be employed. Usually, a density gradient centrifuga-
tion or the swim-up technique is adequate.  

Another significant problem is related to the debris which is often present in the sperm extenders, due 
to the egg yolk or milk proteins. If debris is not removed or gated out, it can lead to false results as foreign 
particles may overlap unstained populations of spermatozoa [4]. On the other hand, there are dyes such as 
tetraethylbenzimidazolylcarbocyanine iodide (JC-1) which are lipophilic and therefore can bind to debris 
and yield misleading results. Gating out debris can be achieved on the FSC vs SSC dot plot, where it usually 
appears to have significantly lower FSC as spermatozoa.  

Mathematical corrections are also possible but are time consuming and difficult to perform.  
Another option is to use an intravital dye, such as Hoechst 33342 which only stains live cells, and there-

fore unstained events can be gated out as debris. The advantage of using this dye also resides in the fact that 
its fluorescent signal (UV range) does not overlap with any of the fluorochromes frequently used for semen 
analysis [5]. Nevertheless, the drawback is that the cytometer must have a violet laser or UV diode. 

3. Instrument setup 
In order to obtain accurate results, the flow cytometer must be checked and properly calibrated on a 

daily basis. Calibration microspheres are usually made of polystyrene latex and are labelled with fluorescent 
dyes. Each laboratory must establish a daily clean-up and calibration protocol, according to the type of 
equipment that is present and the instructions of the producer. Standardization of the procedure is very 
important, as it provides reproducibility, accuracy and reliability of results.  

Next, the machine can be prepared for analysis, by choosing the optimum channels and optical filters 
needed, according to the type of experiment that is required. Usually, the blue 488 nm Argon ion laser at 488 
nm is the only one that is needed, since most of the dyes used for sperm analysis emit green, orange or red 
fluorescence. Green fluorescence is read in FL1, orange in FL2 and red in FL3. Regarding filters, the 530/28 
BP should be used for FL1, the 585/42 BP for FL2 and the 650 LP for FL3. All dot plots or histograms should 
be set to the logarithmic scale and signal height should be acquired for all parameters. 

Compensation should always be performed, whenever multiple dyes are used to stain the same semen 
sample, in order to avoid fluorescence spill over and false results. This is usually achieved using compensa-
tion beads, that are stained with the same fluorochromes as those used in the experiment. Experienced users 
may also choose to perform manual compensation, after the samples are acquired.  

Actual analysis begins by plotting forward scatter height (FSC-H) vs side scatter height (SSC-H) in order 
to define and gate the sperm population. This will also allow to remove from the gate any debris (mostly 
originating from the extenders) and also to eliminate any electronic noise. A total number of at least 10,000 
events should be acquired, and, since the cells (spermatozoa) are small, the sample should be run at low 
speed for better accuracy. 

4. Sperm viability assessment 
DNA intercalating agents are fluorescent molecules, capable of passing through cellular membranes 

of cells and therefore stain the nuclei. Some of the molecules are able to pass through intact membranes and 
therefore also stain the nuclei of live cells. A frequently utilized green fluorescent dye, that stains all nuclei 
of spermatozoa (damaged or intact), is SYBR-14 (maximum emission at 516 nm) which is used in combina-
tion with a red dye - propidium iodide (PI, maximum emission at 617 nm). The latter is also an intercalating 
agent which can only penetrate the damaged plasma membrane and therefore stains the nucleus of only 
dead spermatozoa. PI signal quenches SYBR-14 fluorescence, thus live spermatozoa are stained in green 
while dead spermatozoa are red [6]. Nowadays, there are several live/dead kits available on the marked, 
which allow differentiation between live and dead spermatozoa. Those kits also permit an easy 



Cluj Vet J 2023, vol 28, issue 46  
 

discrimination between spermatozoa and debris, and therefore eliminate the need of performing additional 
staining steps.  

These kits usually contain a 1 mM solution of SYBR-14 in DMSO and a 2.4 mM solution of PI in water. 
A 20 µM SYBR-14 stock solution in DMSO is initially prepared, which can be stored frozen. When needed, 
the working solution is made by adding 5 µL of the 20 µM SYBR-14 solution and 50 µL of the 2.4 mM PI 
solution to 10 ml of buffer, such as HEPES for approximately 20 samples.  

Semen samples can be successfully stained with this working solution, although the manufacturers 
recommend separate incubations. Spermatozoa should be diluted to a concentration of 1-2x106/ml in 0.5 ml 
staining solution, in cytometry tubes. Incubation should be made in the dark at 37°C and run in the cytom-
eter right away. Two dot plots are needed for each tube, one for spermatozoa gating (FSC-H vs SSC-H) and 
another one for viability assessment (FL1-H vs FL3-H). Following the analysis, 3 distinct populations are 
visible on the dot plot:  

• SYBR-14+/PI– (live spermatozoa);  
• SYBR-14+/PI+ (moribund spermatozoa);  
• SYBR-14–/PI+ (dead spermatozoa).  
The SYBR-14–/PI– events are likely to represent debris which should be gated out. 
Alternatively, sperm viability can be assessed using a combination of 3 fluorescent dyes - SNARF-1, 

YO-PRO-1 and ethidium homodimer. Thus, 4 subpopulations of spermatozoa can be detected: one viable, 
with stable membranes (SNARF-1+), and three with compromised membranes: YO-PRO-1+/Eth-, YO-PRO-
1-/Eth+ and YO-PRO-1+/Eth+ [7,8]. 

5. Evaluation of acrosome reaction 
The acrosome is a structure that covers the anterior part of spermatozoa in mammals and contains 

enzymes that allow penetration of the zona pellucida during fertilization. Semen cryopreservation some-
times induces a so-called acrosome reaction, which means inactivation of the specific acrosomal enzymes, 
which renders spermatozoa inefficient [9]. Acrosome integrity can be assessed using FITC or PE labelled 
plant lectins (pea agglutinin-PSA or peanut agglutinin-PNA). PSA cannot breach the membrane of an intact 
acrosome and thus, if stained, spermatozoa are judged as damaged.  In practice, PNA is preferred to PSA 
as the latter was demonstrated to non-specifically bind to the egg-yolk found in semen extenders as well as 
to other fragments of spermatozoa [10]. 

To prepare the stock solution, lyophilized PNA-FITC is resuspended in water to a concentration of 
0.1-1 mg/ml and then stored frozen until use. The 1 mg/ml solution is usually preferred. If storage at the 
refrigeration temperature is needed, a supplementation with 2 mM Na-azide is mandatory. 

The usual protocol when assessing the acrosome reaction is to combine PNA-FITC staining with PI, 
in order to also observe the population of non-viable spermatozoa. The PI stock solution can also be stored 
frozen, at a concentration between 50 µg-5mg/ml, but most frequently a 1 mg/ml solution is preferred.  

The working solutions can easily be prepared based on the stock solutions at 1 mg/ml. After thawing, 
10 µl of both the PNA-FITC and PI solutions are added to 10 ml of PBS and 0.5 ml of the resulting solution 
are used to dilute the semen sample to a concentration of 1-2 x 106 spermatozoa/ml. The working solution 
should be kept in the darkness until use, but not more than 12-24 hours. After staining, spermatozoa should 
be incubated in darkness for 15 minutes at 37°C. 

Next, the samples are run in the flow cytometer and two dot plots are needed: one for spermatozoa 
gating (FSC-H vs SSC-H) and another for acrosome reaction/viability (FL1-H vs FL3-H). 

Following analysis, four different populations of spermatozoa can be identified:  
• the PNA-FITC–/PI– population is represented by viable spermatozoa with unreacted acrosome; 
• the PNA-FITC+/PI– population is represented by viable spermatozoa with reacted acrosome; 
• the PNA-FITC+/PI+ population is represented by moribund or dead spermatozoa with reacted acro-

some;  
• the PNA-FITC–/PI+ population is represented by moribund or dead spermatozoa with unreacted acro-

some. 

6. Evaluation of mitochondrial activity 
In mammalian spermatozoa, mitochondria are essential organelles which play a crucial role for  their 

motility and fertilizing capability, by contributing to ATP and reactive oxygen species  production as well 



Cluj Vet J 2023, vol 28, issue 47  
 

as calcium level control. Their integrity and activity can be assessed by quantitatively evaluating their mem-
brane potential, using a fluorescent dye called 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazoylcarbocy-
anine iodide (JC-1). When mitochondrial membrane potential is high, JC-1 forms combinations that produce 
red fluorescence, while in the case of low mitochondrial membrane potential, JC-1 stays monomeric and 
emits green fluorescence (11).  

The JC-1 stock solution can be prepared by resuspending the lyophilized powder in DMSO, to a con-
centration of 2 mg/ml (3 mM) and stored frozen in a dark vial. The working solution is a 1000 fold dilution 
of the stock solution. Therefore, 10 µl of the stock solution should be added to 10 ml of PBS and 0.5 ml of the 
resulting solution are used to dilute the semen sample to a concentration of 1-2 x 106 spermatozoa/ml. Fol-
lowing incubation for 15-20 minutes in darkness at 37°C, samples are run in the flow cytometer and visual-
ized in two dot plots: one for gating the spermatozoa (the usual FSC-H vs SSC-H) and another for mitochon-
drial membrane potential (FL1-H vs FL2-H). 

Analysis of dot plots allows classification of ejaculates according to mitochondrial activity of sperma-
tozoa, as follows:  

• spermatozoa with high FL1-H and low FL2-H have a low mitochondrial membrane potential 
and are considered of low fertilizing ability; 

• spermatozoa with low FL1-H and high FL2-H have high mitochondrial membrane potential 
and potentially poses a fertilizing ability (this category should be the most abundant in good 
quality ejaculates); 

• spermatozoa with high FL1-H and high or moderate FL2-H are considered to have large gaps 
in their mitochondria and are therefore of lower quality; 

• spermatozoa with low FL1-H and low FL2-H are likely dead and have a damaged midpiece. 

7. Conclusions 
Flow cytometry is an extremely useful and powerful tool that can be used for advanced semen anal-

ysis as it provides quick and reliable results, enabling an accurate estimation of various semen parameters.  
Together with computer assisted sperm analysis CASA systems, flow cytometers allow an in-depth 

analysis of fresh, chilled or frozen/thawed semen, which is currently essential for research purposes, but also 
for diagnosis of various male-related fertility disorders, both in veterinary and human medicine.  

The equipment required is indeed quite expensive and the operators need special training, while ex-
perience is also an asset. 

 
Author Contributions: DC, BȚ, VT and MC: writing—original draft preparation, JTB and VG: writing—review and 
editing. All authors have read and agreed to the published version of the manuscript”. 

Funding: DC, JTB, VG BȚ, VT and MC were funded by an International Collaborative Grant of the European Economic 
Space between Romania, Iceland, and Norway 2014–2021: “Continuous Flow Interchange of Communication and 
Knowledge in Biomedical University Research—FLOW”, No. 21-COP-0034.  

Conflicts of Interest: The authors declare no conflict of interest. 

References 
 

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integrity of dog spermatozoa and for evaluation of different methods of cryopreservation. J Reprod Fertil 2001, Suppl 57, 
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	1. Introduction
	2. Semen Sample Preparation
	3. Instrument setup
	4. Sperm viability assessment
	5. Evaluation of acrosome reaction
	6. Evaluation of mitochondrial activity
	7. Conclusions
	References

