Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm.
Abstract: An acrosomal staining technique that can differentiate between living and dead sperm was developed for equine sperm. The fluoresceinated lectin Pisum sativum agglutinin (FITC-PSA) was used to identify the presence or absence of acrosomal contents, while the supravital nuclear dye Hoechst 33258 (H258) was used to assess viability. The accuracy of the FITC-PSA acrosomal stain was tested by comparing the percentage of sperm that had lost their acrosomal contents, detected by the staining method, with that detected by transmission electron microscopy (TEM). Following capacitation in vitro, the acrosomal status of sperm induced to acrosome react with A23187 and of control sperm were very similar with the staining technique and TEM, confirming the accuracy of the FITC-PSA acrosomal stain. We investigated the relationship between viability as measured by exclusion of H258 and motility as measured by three methods: one subjective and two objective. Although there was a good correlation between viability and motility as measured by all three methods (r = 0.88, 0.85, 0.75), there was always a proportion of viable sperm that were nonmotile. The physiology of the viable, nonmotile sperm was further investigated by comparing for individual sperm the viability as measured by exclusion of H258 with the mitochondrial function as measured by rhodamine 123. A good correlation (r = 0.99) was found to exist between viability and mitochondrial function, indicating that viable, nonmotile sperm possess functional mitochondria and confirming the ability of supravital staining to distinguish between living and dead sperm. We determined that 29-81% of the sperm in semen that had lost their acrosomal contents were in fact dead. Thus, this acrosomal staining technique can provide more relevant endpoints for future investigations of capacitation, the acrosome reaction, and sperm handling techniques in the horse.
Publication Date: 1993-07-01 PubMed ID: 7693637
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research paper discusses the development and validation of a staining technique, designed for equine sperm, that can differentiate between live and dead sperm. It demonstrates the accuracy and efficiency of the method, and explores its potential applications within equine fertility studies and sperm handling techniques.
Acrosomal Staining Technique
- The main focus of the paper is the development and validation of an acrosomal staining method that differentiates between living and dead equine sperm.
- This method utilises two dyes – the fluoresceinated lectin Pisum sativum agglutinin (FITC-PSA) and the supravital nuclear dye Hoechst 33258 (H258). FITC-PSA aids in ascertaining the presence or absence of acrosomal contents. H258, meanwhile, helps in assessing viability.
- The accuracy of the FITC-PSA acrosomal stain is evaluated by comparing the detected percentages of sperm that had lost their acrosomal contents with results from transmission electron microscopy (TEM) – a different, more established method.
Confirmation of Staining Technique Accuracy
- The paper reports that the acrosomal status ascertained via staining methods was closely similar to that determined through TEM, following a process known as in vitro capacitation. This confirmed the accuracy of the FITC-PSA acrosomal stain.
- The resistance of sperm to the penetration of H258 was used to measure viability, and this was compared with motility measured by three different methods. Despite a good correlation, some sperm were found to be viable but nonmotile.
Investigation of Viable, Non-motile Sperm
- To further explore this phenomenon, individual viable, nonmotile sperm were studied. Viability was again judged by resistance to H258 penetration, while mitochondrial function was assessed by using rhodamine 123.
- This comparison showed a strong correlation between viability and mitochondrial function, which indicates that even nonmotile sperm can have functional mitochondria. This finding solidified the capability of the staining technique in distinguishing between living and dead sperm.
Semen Analysis and Future Application
- The authors determined that 29-81% of sperm that had lost their acrosome content were dead, based on semen analysis. This provides a more precise evaluation of sperm viability than was previously available.
- This acrosomal staining method offers potential for more detailed analysis in future studies of capacitation, the acrosome reaction, and sperm handling techniques in horses.
Cite This Article
APA
Casey PJ, Hillman RB, Robertson KR, Yudin AI, Liu IK, Drobnis EZ.
(1993).
Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm.
J Androl, 14(4), 289-297.
Publication
Researcher Affiliations
- Department of Reproduction, School of Veterinary Medicine, University of California, Davis 95616.
MeSH Terms
- Acrosome / chemistry
- Acrosome / physiology
- Acrosome / ultrastructure
- Animals
- Bisbenzimidazole / analysis
- Bisbenzimidazole / standards
- Calcimycin / pharmacology
- Cell Death / physiology
- Cell Separation / methods
- Cell Separation / standards
- Fluorescein-5-isothiocyanate / analysis
- Fluorescein-5-isothiocyanate / standards
- Horses / physiology
- Infertility, Male / diagnosis
- Infertility, Male / physiopathology
- Lectins / analysis
- Lectins / standards
- Male
- Microscopy, Electron / methods
- Mitochondria / chemistry
- Mitochondria / physiology
- Mitochondria / ultrastructure
- Plant Lectins
- Reproducibility of Results
- Rhodamine 123
- Rhodamines
- Sperm Motility / physiology
- Spermatozoa / cytology
- Spermatozoa / physiology
- Spermatozoa / ultrastructure
- Staining and Labeling / standards
Citations
This article has been cited 6 times.- Alkhawagah AR, Ricci A, Banchi P, Martino NA, Poletto ML, Donato GG, Nervo T, Vincenti L. Effect of Epidermal Growth Factor (EGF) on Cryopreserved Piedmontese Bull Semen Characteristics. Animals (Basel) 2022 Nov 17;12(22).
- Aguiar GB, Caldas-Bussiere MC, Maciel VL Jr, de Carvalho CSP, de Souza CLM. Association of L-arginine with heparin on the sperm capacitation improves in vitro embryo production in bovine. Anim Reprod 2019 Nov 18;16(4):938-944.
- Chaveiro A, Cerqueira C, Silva J, Franco J, Moreira da Silva F. Evaluation of frozen thawed cauda epididymal sperms and in vitro fertilizing potential of bovine sperm collected from the cauda epididymal. Iran J Vet Res 2015 Spring;16(2):188-93.
- Carreira JT, Mingoti GZ, Rodrigues LH, Silva C, Perri SH, Koivisto MB. Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa. Acta Vet Scand 2012 Jan 12;54(1):1.
- Sutkeviciene N, Riskeviciene V, Januskauskas A, Zilinskas H, Andersson M. Assessment of sperm quality traits in relation to fertility in boar semen. Acta Vet Scand 2009 Dec 16;51(1):53.
- Katila T. In vitro evaluation of frozen-thawed stallion semen: a review. Acta Vet Scand 2001;42(2):199-217.
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