Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

Abstract

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of 'bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity — A flow cytometric study

“…Moreover, mitochondria have an important role in the maintenance of Ca 2+ -homeostasis -the mitochondrial membrane depolarization causes decreased Ca 2+ influx [20]. This latter and the increase in intracellular Ca 2+ level are key components of fish sperm activation [15] and necessary for capacitation in mammalian spermatozoa [14].…”

Assessment of Ageing-Dependent Effects on Sperm Functions following Semen Cryopreservation

“…4 Moreover, the sperm membranes are an integral part of the acrosome reaction and are involved in the fertilizationrelated events, so deterioration in the structural and functional integrity of spermatozoa is an obvious sign of the ageing processes, as is evident after cryopreservation. 5 Assessment of the cryo-induced sperm ageing processes, using a combination of different fluorescent probes (lipophilic cationic JC-1 in combination with propidium iodide -PI, JC-1/PI; rhodamine 123, R123/PI; MitoTracker Green, MTG; SYBR-14/PI; carboxyfluorescein diacetate, CFDA/PI; Hoechst 33258, H258; fluorescein isothiocyanate (FITC)-labeled peanut agglutinin, FITC-PNA/PI; FITC-labeled Pisum sativum agglutinin, FITC-PSA/PI), has provided more detailed information on several sperm attributes that are required to identify individuals with poor and good semen freezability. 1,5 Furthermore, the ageing-associated decrease in post-thaw sperm viability is concomitant with an increase in lipid peroxidation measured by malondialdehyde (MDA) production, capacitation-like destabilization of sperm membranes (antibiotic chlortetracycline, CTC; Merocyanine 540, M540), apoptotic sperm cells (Annexin-V/PI; Yo-Pro-1/ PI; caspase activation) or sperm DNA fragmentation (Comet assay, sperm chromatin structure assay, SCSA; terminal deoxynucleotidyl transferase-mediated dUDP nick end labelling assay, TUNEL; sperm chromatin dispersion assay), which significantly compromises the fertilizing ability of frozen-thawed spermatozoa.…”

“…4,5 Despite the effectiveness of these sperm assays, the underlying mechanisms involved in the cryo-induced ageing-dependent changes, within different compartments of the spermatozoon, are still poorly understood.…”

“…1 A plethora of sperm markers have provided evidence of the ageingdependent effects on sperm cryo-survival. 5 Accordingly, molecular markers, used to detect the sperm ageing-dependent processes associated with the freezing-thawing procedure, have made possible a more widespread analysis of different sperm attributes that are considered as a prerequisite for successful fertilization. 6 Cryo-induced sperm ageing processes have been manifested in compromised sperm motility, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and acrosome membrane integrity (AMI), which are the key determinants of semen quality.…”

“…3 It is noteworthy that ageing-dependent processes in frozen-thawed semen occur in different sperm compartments, and are associated with membrane modifications of specific lipid-protein interactions, activation of an apoptosis-like mechanism and reduced genomic integrity, which consequently compromise sperm cryo-survival. 4,5 Moreover, ageing is a natural process occurring in sperm cells during semen preservation, and there is no single sperm test that can effectively predict the fertilizing capacity of semen following the different reproductive technologies. 6 Cryo-induced ageing-dependent processes in spermatozoa have marked effects on different sperm attributes, either directly or indirectly, resulting in impaired fertilizing ability of the post-thaw semen.…”