A fast, low-cost and efficient method for the diagnosis of sperm DNA fragmentation in several species.
Abstract: Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high-precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue-stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.
© 2017 Blackwell Verlag GmbH.
Publication Date: 2017-10-30 PubMed ID: 29086436DOI: 10.1111/rda.13087Google Scholar: Lookup
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- Journal Article
Summary
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The study is about a new efficient method for diagnosing a condition called sperm DNA fragmentation in various animal species. This novel technique uses a stain called toluidine blue (TB), which proves to be faster and less expensive than existing methods.
Background of the Study
- The research focuses on sperm DNA fragmentation, an issue that affects reproductive efficiency in various species.
- Different techniques are available to assess sperm DNA integrity, such as the Alkaline Comet, TUNEL, and Sperm Chromatin Structure Assay.
- These existing methods are often labor-intensive and necessitate high-precision equipment, making them time-consuming and expensive.
Purpose of the Study
- The study aimed to standardize a new, efficient, and cost-effective method for analyzing DNA fragmentation of spermatozoa (sperm cells) in various species: dogs, cats, bulls, rams, and stallions.
- The new technique involved the use of toluidine blue (TB), a type of staining substance.
Methodology
- Six animals from each species, all in reproductive age, were used in the study.
- The sperm was collected differently for every species, based on their unique characteristics. It was divided into two parts: one kept at 5°C (intact DNA sample) and the other was exposed to UV light for 4 hours to induce DNA fragmentation.
- Later, the samples were combined to obtain varying proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%).
- Semen smears were created and subjected to TB staining. Spermatozoa stained blue were identified as having DNA fragmentation.
Findings
- The results showed strong linear regression coefficients between the expected proportion of damaged DNA and the outcomes of the TB staining for all the species.
- The toluidine blue (TB) stain method was established as a quick and effective technique for studying sperm DNA fragmentation.
Cite This Article
APA
Rui BR, Angrimani D, Bicudo LC, Losano J, Nichi M, Pereira R.
(2017).
A fast, low-cost and efficient method for the diagnosis of sperm DNA fragmentation in several species.
Reprod Domest Anim, 53(1), 171-175.
https://doi.org/10.1111/rda.13087 Publication
Researcher Affiliations
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
MeSH Terms
- Animals
- Cats / genetics
- Cattle / genetics
- DNA Fragmentation / radiation effects
- Dogs / genetics
- Horses / genetics
- Male
- Sheep / genetics
- Spermatozoa
- Staining and Labeling / methods
- Staining and Labeling / veterinary
- Tolonium Chloride / chemistry
- Ultraviolet Rays / adverse effects
Citations
This article has been cited 6 times.- Flores RB, Angrimani DSR, Brito MM, de Almeida LL, Lopes JVM, Losano JDA, Vannucchi CI. Frozen-Thawed Sperm Analysis of Benign Prostatic Hyperplasia Dogs Treated With Finasteride.. Front Vet Sci 2022;9:901943.
- Schäfer-Somi S, Colombo M, Luvoni GC. Canine Spermatozoa-Predictability of Cryotolerance.. Animals (Basel) 2022 Mar 15;12(6).
- Al-Khaldi K, Yimer N, Sadiq MB, Firdaus Jesse Bin Abdullah F, Salam Babji A, Al-Bulushi S. Edible bird's nest supplementation in chilled and cryopreserved Arabian stallion semen.. Saudi J Biol Sci 2022 Mar;29(3):1577-1584.
- Angrimani DSR, Bicudo LC, Llamas Luceño N, Rui BR, Silva MF, Losano JDA, Leemans B, Van Soom A, Vannucchi CI. Does finasteride treatment for benign prostatic hyperplasia influence sperm DNA integrity in dogs?. Basic Clin Androl 2020;30:9.
- Barbosa BS, Dos Santos FA, do Macêdo LB, Izzo RG, Fernandes DP, Praxedes ÉA, Silva AR, Bezerra MB. Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa.. Anim Reprod 2020 Jan 30;17(1):e20190067.
- Yildiz K, Yuksel S. Use of microfluidic sperm extraction chips as an alternative method in patients with recurrent in vitro fertilisation failure.. J Assist Reprod Genet 2019 Jul;36(7):1423-1429.
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