Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.
Abstract: Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.
© 2013 Blackwell Verlag GmbH.
Publication Date: 2013-07-24 PubMed ID: 23889365DOI: 10.1111/rda.12221Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article explores the use of Trypan blue/Giemsa staining to evaluate the quality and fertilizing ability of sperm in Salernitano stallions, and how these factors relate to pregnancy rates in mares.
Background and Objectives
- The aim was to assess the functionality of Trypan blue/Giemsa staining as a tool for evaluating sperm membrane integrity, acrosomal intactness, and morphology in Salernitano stallions.
- This assessment would also determine the correlation of sperm quality to pregnancy rates in mares. Consequently, the possibility of employing this method as a practical tool to predict fertilizing ability in vitro was examined.
Methodology
- The researchers collected forty-one ejaculates from three stallions of the Salernitano Horse Breed.
- These were evaluated for parameters such as gross appearance, volume, visual motility, and membrane integrity using Trypan blue/Giemsa staining.
- During the breeding season (April to July), thirty-five mares were inseminated with the collected ejaculates.
Results
- Significant differences were found among stallions in terms of volume, sperm concentration (p < 0.05), and visual motility (p < 0.01).
- Sperm motility, concentration, and the total number of sperm decreased in June-July (p < 0.01).
- The numbers of live sperm with intact acrosomes (LSIA) and proximal droplets (PD) were lower in June-July, while the percentage of acrosome-reacted sperm (ARS) increased (p < 0.05).
- No differences in fertility were observed among the stallions, with an average per-cycle fertility of 44.6%, and a pregnancy rate of 68.6%.
- Ejaculates used to inseminate mares that became pregnant had a higher percentage of LSIA compared to those used in mares which did not become pregnant (p < 0.05).
Conclusions
- The Trypan blue/Giemsa staining method provided reliable results, reinforcing its significance as a test variable for sperm membrane integrity.
- Receiver operating characteristic (ROC) analysis confirmed this reliability and determined the sensitivity of the test as 85% at a cut-off value of 48% LSIA. This suggests that the staining can accurately identify low-quality ejaculates.
- In conclusion, the Trypan blue-Giemsa staining method proved to be an effective means to evaluate sperm membrane integrity and could potentially be applied in the field to predict sperm fertilizing ability.
Cite This Article
APA
Serafini R, Longobardi V, Spadetta M, Neri D, Ariota B, Gasparrini B, Di Palo R.
(2013).
Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.
Reprod Domest Anim, 49(1), 41-47.
https://doi.org/10.1111/rda.12221 Publication
Researcher Affiliations
- Department of Veterinary Medicine and Animal Production, Federico II University of Naples-Via F., Naples, Italy.
MeSH Terms
- Acrosome / ultrastructure
- Acrosome Reaction
- Animals
- Azure Stains
- Breeding
- Cell Membrane / ultrastructure
- Female
- Fertility
- Horses
- Insemination, Artificial / veterinary
- Male
- Pregnancy
- Pregnancy Rate
- ROC Curve
- Sensitivity and Specificity
- Sperm Count / veterinary
- Sperm Motility
- Spermatozoa / physiology
- Spermatozoa / ultrastructure
- Staining and Labeling / methods
- Staining and Labeling / veterinary
- Trypan Blue
Citations
This article has been cited 10 times.- Laoung-On J, Jaikang C, Saenphet K, Sudwan P. Effect of Nelumbo nucifera Petals Extract on Antioxidant Activity and Sperm Quality in Charolais Cattle Sperm Induced by Mancozeb. Plants (Basel) 2022 Feb 26;11(5).
- Kumari S, Tevatiya S, Rani J, Das De T, Chauhan C, Sharma P, Sah R, Singh S, Pandey KC, Pande V, Dixit R. A testis-expressing heme peroxidase HPX12 regulates male fertility in the mosquito Anopheles stephensi. Sci Rep 2022 Feb 16;12(1):2597.
- Momeni HR, Abnosi MH, Eskandari N. Quantitative evaluation of human sperm viability using MTT assay: A laboratory study. Int J Reprod Biomed 2020 Nov;18(11):983-988.
- Scalisi EM, Pecoraro R, Salvaggio A, Corsaro A, Messina G, Ignoto S, Lombardo BM, Brundo MV. Evaluation of dimethoate toxicity on fertilization and on embryonic development of Paracentrotus lividus (Lamarck, 1816). Toxicol Res (Camb) 2020 Jul;9(4):537-543.
- Dong YS, Hou WG, Li Y, Liu DB, Hao GZ, Zhang HF, Li JC, Zhao J, Zhang S, Liang GB, Li W. Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells. Cell Death Differ 2016 May;23(5):787-800.
- Khalil WA, Mostafa HE, Derbala MK, Alfattah MA, Alhujaili W, Hassan MAE, El-Harairy MA, Abdelnour SA. Efficacy of butylated hydroxytoluene nanoparticles in enhancing the quality and preservation of stallion chilled semen. Vet Res Commun 2025 Dec 3;50(1):62.
- Wei P, Wang N, Zhang Q, Wang W, Sun H, Liu Z, Yan T, Wang Q, Qiu L. Nano-ZnO-modified hydroxyapatite whiskers with enhanced osteoinductivity for bone defect repair. Regen Biomater 2024;11:rbae051.
- Ghanbari A, Jalili C, Shahveisi K, Akhshi N. Harmine exhibits anti-apoptotic properties and reduces diabetes-induced testicular damage caused by streptozotocin in rats. Clin Exp Reprod Med 2024 Dec;51(4):324-333.
- Fan C, Aihemaiti A, Fan A, Dilixiati A, Zhao X, Li Z, Chen C, Zhao G. Study on the correlation of supplementation with L-citrulline on the gastrointestinal flora and semen antifreeze performance of ram. Front Microbiol 2024;15:1396796.
- Agudo-Rios C, Rogers A, King I, Bhagat V, Nguyen LMT, Córdova-Fletes C, Krapf D, Strauss JF 3rd, Arévalo L, Merges GE, Schorle H, Roldan ERS, Teves ME. SPAG17 mediates nuclear translocation of protamines during spermiogenesis. Front Cell Dev Biol 2023;11:1125096.
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