Comparison of methods for assessing integrity of equine sperm membranes.
Abstract: Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = -3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = -11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.
Copyright © 2011 Elsevier Inc. All rights reserved.
Publication Date: 2011-04-14 PubMed ID: 21496902DOI: 10.1016/j.theriogenology.2011.02.012Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study investigates the best method for assessing the integrity of horse sperm membranes, an important factor for evaluating sperm quality in stallions. Three methods were compared: flow cytometry using a specific type of stain, using an automated cell counting device with a stain, and the eosin-nigrosin stain method.
Study Methods
The researchers method included three processes:
- They subjected raw horse semen to different treatments that contained 20 to 80% seminal plasma in extender. These treatments simulated spontaneous loss of sperm membrane integrity (SMI).
- The SMI was evaluated immediately after the treatment, and then after one day and two days of cooled storage.
- The agreement between the three methods was determined using a statistical technique called the Bland-Altman methodology.
Results
Research results indicated that:
- Eosin-nigrosin staining provided slightly higher overall mean values for SMI than the flow cytometry method did.
- Flow cytometry gave slightly higher overall mean values for SMI compared to the automated cell counter.
- The agreement between SMI measurements from the different methods decreased as the percentage of sperm with damaged membranes increased.
Comparing the Ranges
The Bland-Altman analysis was carried out to compare the different methods’ agreement:
- When the semen had 50-79% membrane-intact sperm, flow cytometry and eosin-nigrosin staining had a greater range of difference in results than flow cytometry and the automated cell counter.
- When sperm populations contained less than 50% membrane-intact sperm, the same pattern was noticed.
Conclusion
The study concluded that eosin-nigrosin staining’s assessments of percentage of membrane-intact sperm agreed less with flow cytometry when fewer than 80% of sperm had intact membranes. The automated cell counter’s assessments of the percentage of membrane-intact sperm agreed less with flow cytometry when fewer than 30% of sperm had intact membranes. The implications of these findings suggest differences in measuring SMI between the methods and the need for further research to optimize these methods.
Cite This Article
APA
Foster ML, Love CC, Varner DD, Brinsko SP, Hinrichs K, Teague S, Lacaze K, Blanchard TL.
(2011).
Comparison of methods for assessing integrity of equine sperm membranes.
Theriogenology, 76(2), 334-341.
https://doi.org/10.1016/j.theriogenology.2011.02.012 Publication
Researcher Affiliations
- Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, USA.
MeSH Terms
- Aniline Compounds
- Animals
- Cell Membrane / physiology
- Cell Membrane / ultrastructure
- Coloring Agents
- Eosine Yellowish-(YS)
- Flow Cytometry / veterinary
- Fluorescent Dyes
- Horses
- Male
- Organic Chemicals
- Propidium
- Sperm Count
- Spermatozoa / ultrastructure
- Staining and Labeling / veterinary
Citations
This article has been cited 2 times.- Macêdo IN, Arruda LCP, de Santana BB, de Moura TCM, Guerra MMP, Bezerra DG, Carneiro GF, Silva SV. The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation.. Anim Reprod 2021;18(4):e20210075.
- Wysokińska A, Kondracki S, Iwanina M. The Usefulness of Selected Physicochemical Indices, Cell Membrane Integrity and Sperm Chromatin Structure in Assessments of Boar Semen Sensitivity.. Asian-Australas J Anim Sci 2015 Dec;28(12):1713-20.
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