Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine ( Canis lupus familiaris) and stallion ( Equus caballus).
Abstract: Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content of carbonyl groups in the SP was measured on the basis of the reactions with DNPH. The following reagent variants were used to dissolve protein precipitates: Variant 1 (V1) - 6M Guanidine solution and Variant 2 (V2) - 0.1M NaOH solution. It has been shown that to obtain reliable results for the measurement of protein carbonylated groups in the dog and horse SP, both 6M Guanidine and 0.1M NaOH may be used. A correlation was also found between the number of carbonyl groups and the total protein content in the canine (V1: r = -0.724; V2: r = -0.847) and stallion (V1: r = -0.336; V2: r = -0.334) SP. Additionally, the study showed a higher content (p≤0.05) of protein carbonyl groups in the stallion SP in the non-breeding season compared to the breeding season. The method based on the reaction with DNPH, due to its simplicity and cost effectiveness, appears to be suitable for large-scale application in the determination of the SP proteins oxidative damage in dog and horse semen.
Copyright© by the Polish Academy of Sciences.
Publication Date: 2023-06-30 PubMed ID: 37389438DOI: 10.24425/pjvs.2023.145020Google Scholar: Lookup
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Summary
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The research studied how 2,4-dinitrophenylhydrazine (DNPH) method can be used to measure oxidative damage in proteins found in dog and horse semen. Findings conclude that it can provide reliable results, and could be applied to assess fertility ability in these animals.
Research Background and Aim
- This research investigates the potential use of 2,4-dinitrophenylhydrazine (DNPH) method in measuring the degree of oxidative damage in seminal plasma (SP) proteins.
- SP proteins contribute to sperm quality in animals, hence determining their oxidative damage is crucial for assessing semen’s fertilizing capability. The goal of this research was to test the DNPH method’s efficacy for this purpose in dogs and horses.
Methodology
- Researchers collected ejaculate samples from eight English Springer Spaniels and seven half-blood stallions. The stallions’ samples were obtained during both breeding and non-breeding seasons.
- A protein carbonyl derivatives measurement technique using DNPH was employed on the SP in the samples. The carbonyl group content in the SP was determined based on its reactions with DNPH.
- Different solvents were used to dissolve protein precipitates: 6M Guanidine solution (Variant 1- V1) and 0.1M NaOH solution (Variant 2 – V2).
Results
- The study found that both 6M Guanidine and 0.1M NaOH can yield reliable results when measuring protein carbonylated groups in canine and equine SP.
- There was a correlation observed between the count of carbonyl groups and the total protein content in both canine and stallion SP, with correlation coefficients for V1 and V2 specified.
- Another noteworthy finding was the higher amount of protein carbonyl groups in the SP of stallions during the non-breeding season compared to the breeding season, which was statistically significant (p≤0.05).
Conclusions
- The method that makes use of the reaction with DNPH was noted to be effective, simple, and economical. Due to these advantages, it shows potential suitability for larger-scale application in determining oxidative damage in SP proteins in dog and horse semen.
Cite This Article
APA
Mogielnicka-Brzozowska M, Woźniak MJ, Cichowska AW, Fraser L, Kraziński B, Strzezek R, Zielińska D.
(2023).
Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine ( Canis lupus familiaris) and stallion ( Equus caballus).
Pol J Vet Sci, 26(2), 177-184.
https://doi.org/10.24425/pjvs.2023.145020 Publication
Researcher Affiliations
- Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957 Olsztyn, Poland.
- Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957 Olsztyn, Poland.
- Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957 Olsztyn, Poland.
- Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957 Olsztyn, Poland.
- Department of Human Histology and Embryology, School of Medicine, Collegium Medicum, University of Warmia and Mazury in Olsztyn, Warszawska 30, 10-082 Olsztyn, Poland.
- Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-957 Olsztyn, Poland.
- Department of Chemistry, Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn, Oczapowskiego 8, 10-719 Olsztyn, Poland.
MeSH Terms
- Male
- Animals
- Horses
- Dogs
- Semen
- Wolves
- Sodium Hydroxide
- Guanidine
- Guanidines
- Oxidative Stress
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