Flow cytometry in assessment of sperm integrity and functionality – a review

Spermatozoa must possess many attributes to fertilize an egg but few laboratory methods can assess all of these attributes simultaneously and objectively. Most laboratory methods used to assess the quality of semen in veterinary andrology can be inaccurate and time-consuming. Laboratory techniques which evaluate only one sperm attribute, often provide results that have a weak correlation with fertility. Therefore, semen used for artificial insemination should not be assessed based on the results of one method only but rather on the comprehensive results of several laboratory tests. Flow cytometry is a modern method of analysing diJ169fferent types of cells, including sperm cells. It is based on the scattering of light and fluorescence, the outcome of which yields results that are recorded and evaluated by computer technology which makes an objective assessment. Flow cytometry in combination with fluorescence microscopy and fluorescent probes provides a comprehensive, accurate, objective, and rapid analysis of the ejaculate. In a short time frame it allows us to test thousands of sperm for their structure and properties, even with a minimal amount of semen. This method enables the evaluation of several indicators simultaneously in a population of sperm as a whole or for each sperm individually. It informs us about the selected indicators of sperm quality in the sample by examining the membrane integrity, DNA, mitochondria, acrosome, oxidative stress, and other properties. Flow cytometry has become an important method of evaluating the functional and morphological properties of sperm and is used for research in veterinary science as well as for a routine assessment of the semen quality.

[1]  P. Blondin,et al.  Bovine Semen Quality Control in Artificial Insemination Centers , 2021, Bovine Reproduction.

[2]  Cathleen A. Towey.  Flow , 2000, My Wilderness.

[3]  M. Dode,et al.  Techniques for sperm evaluation using fluorescent probes , 2015 .

[4]  Masashi Sekino,et al.  Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement , 2015, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[5]  F. Peña.  Multiparametric flow cytometry: a relevant tool for sperm function evaluation , 2015 .

[6]  Greg Finak,et al.  Critical assessment of automated flow cytometry data analysis techniques , 2013, Nature Methods.

[7]  Christopher J Chang,et al.  Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. , 2012, Nature chemistry.

[8]  T. Kalina,et al.  EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols , 2012, Leukemia.

[9]  P. Sutovsky,et al.  Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization. , 2012, International journal of andrology.

[10]  J. Roseiro,et al.  Applications and perspectives of multi-parameter flow cytometry to microbial biofuels production processes. , 2012, Trends in biotechnology.

[11]  H. Bollwein,et al.  Osmotic properties of stallion sperm subpopulations determined by simultaneous assessment of cell volume and viability. , 2011, Theriogenology.

[12]  A M Petrunkina,et al.  Cytometric solutions in veterinary andrology: Developments, advantages, and limitations , 2011, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[13]  Szabolcs Nagy,et al.  Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. , 2011, Asian journal of andrology.

[14]  E. Celeghini,et al.  Damage assessment of the equine sperm membranes by fluorimetric technique , 2010 .

[15]  P. de Paz,et al.  Probes and techniques for sperm evaluation by flow cytometry. , 2010, Reproduction in domestic animals = Zuchthygiene.

[16]  Suehee Kim,et al.  Apoptosis-like change, ROS, and DNA status in cryopreserved canine sperm recovered by glass wool filtration and Percoll gradient centrifugation techniques. , 2010, Animal reproduction science.

[17]  A. Giwercman,et al.  Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case-control study. , 2010, International journal of andrology.

[18]  P. Lybaert,et al.  Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa. , 2009, Histology and histopathology.

[19]  A. J. Soler,et al.  See Blockindiscussions, Blockinstats, Blockinand Blockinauthor Blockinprofiles Blockinfor Blockinthis Blockinpublication Sperm Blockincharacteristics Blockinand Blockinin Blockinvitro Blockinfertilization Ability Blockinof Blockinthawed Blockinspermatozoa Blockinfrom Blockinblack Manchega Blockinram , 2022 .

[20]  H. Rodríguez-Martínez,et al.  Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes. , 2009, Reproduction.

[21]  A. Agarwal,et al.  The diagnostic and therapeutic applications of flow cytometry in male infertility , 2009 .

[22]  R. Aitken,et al.  Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. , 2008, The Journal of clinical endocrinology and metabolism.

[23]  J. Ballescà,et al.  Human sperm DNA fragmentation: Correlation of TUNEL results as assessed by flow cytometry and optical microscopy , 2007, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[24]  B. Ball,et al.  Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium. , 2007, Theriogenology.

[25]  J. Lornage,et al.  Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome. , 2007, Fertility and sterility.

[26]  R. Pérez-Pé,et al.  Comparative study of four different sperm washing methods using apoptotic markers in ram spermatozoa. , 2006, Journal of andrology.

[27]  D. Evenson,et al.  Clinical aspects of sperm DNA fragmentation detection and male infertility. , 2006, Theriogenology.

[28]  M. Spanô,et al.  Flow cytometry applications in the evaluation of sperm quality: semen analysis, sperm function and DNA integrity. , 2005, Contraception.

[29]  Hongtao Zhao,et al.  Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[30]  G. Evans,et al.  Flow cytometric evaluation of sperm parameters in relation to fertility potential. , 2005, Theriogenology.

[31]  P. James,et al.  Lipid diffusion in sperm plasma membranes exposed to peroxidative injury from oxygen free radicals , 2004, Molecular reproduction and development.

[32]  M. Melamed,et al.  Comparison of human and mouse sperm chromatin structure by flow cytometry , 2004, Chromosoma.

[33]  J. Brouwers,et al.  In situ detection and localization of lipid peroxidation in individual bovine sperm cells. , 2003, Free radical biology & medicine.

[34]  H. Rodríguez-Martínez.  Laboratory semen assessment and prediction of fertility: still utopia? , 2003, Reproduction in domestic animals = Zuchthygiene.

[35]  S. Nagy,et al.  A Triple-Stain Flow Cytometric Method to Assess Plasma- and Acrosome-Membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-Yolk Particles1 , 2003, Biology of reproduction.

[36]  C. Peterson,et al.  SPERM DNA FRAGMENTATION IS INCREASED IN COUPLES WITH UNEXPLAINED RECURRENT PREGNANCY LOSS , 2003, Archives of andrology.

[37]  J. Hendry,et al.  Germ Cell and Dose-Dependent DNA Damage Measured by the Comet Assay in Murine Spermatozoaa after Testicular X-Irradiation1 , 2002, Biology of reproduction.

[38]  M. Buhr,et al.  Sperm Apoptosis in Fresh and Cryopreserved Bull Semen Detected by Flow Cytometry and Its Relationship with Fertility1 , 2002, Biology of reproduction.

[39]  TsuyoshiKasai,et al.  Relationship between sperm mitochondrial membrane potential,sperm motility,and fertility potential , 2002 .

[40]  J. Fuhrman,et al.  Significance of Size and Nucleic Acid Content Heterogeneity as Measured by Flow Cytometry in Natural Planktonic Bacteria , 1999, Applied and Environmental Microbiology.

[41]  M. Vidament,et al.  New staining methods for sperm evaluation estimated by microscopy and flow cytometry. , 1997, Theriogenology.

[42]  R. Haugland,et al.  Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. , 1994, Journal of andrology.

[43]  D. Evenson,et al.  Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry. , 1990, Journal of dairy science.