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)

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 applicabili-ty 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 & LE;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 applica-tion in the determination of the SP proteins oxidative damage in dog and horse semen.

Automated summary

The study aimed to verify the applicability of using DNPH to measure protein carbonyl derivatives in canine and stallion seminal plasma. The content of carbonyl groups in the seminal plasma was measured, and it was found that both 6M Guanidine and 0.1M NaOH could be used to obtain reliable results. Additionally, a higher content of protein carbonyl groups was found in the stallion seminal plasma during the non-breeding season compared to the breeding season.