Enlarged spectrum of seminological diagnoses using the resazurin colour reaction; a spectrophotometric application

Abstract

Two sets of investigations were carried out in the present study to establish whether, in semen samples processed for the resazurin reaction (RT), the colour change and its intensity were informative for both the quality and quantity of spermatozoa. First, the results of RT were read visually using single semen samples from 42 consecutive men from infertile couples. In the second step, the results were additionally read using a spectrophotometer. Single semen samples obtained from another 87 consecutive men from infertile couples and 12 men from fertile couples were analysed in this step. Visual analysis, which involved grading against and RT colour chart, revealed a significant positive correlation between the RT results and the quantity and quality of spermatozoa. Azoospermia was diagnosed by grade number 1 (dark purple colour), while the highest grades 8-11 (colours dark red to pink) represented a homogeneous group of patients with the highest quality of spermatozoa (> 60% motility) and quantity (> 60 x 10(6)/ml ejaculate). Normal parameters of spermatozoa, according to WHO, appeared with a high predominance (89-100% of cases) in this group and were present in most of the samples. Thus, the grades 8-11 might represent semen samples with optimal fertility potential. Spectrophotometric analysis of RT revealed that visual characteristics corresponded closely to the values for optical density (OD) of the samples. The OD for normozoospermic samples ranged between 1.48 and 1.84; for oligozoospermia between 1.2 and 1.48; for azoospermia between 0.98 and 1.1. The OD for samples with putative optimal fertility potential ranged between 1.6 and 1.84. RT evaluation using a spectrophotometer may therefore provide a tool for obtaining a wider spectrum of seminological diagnoses than classic microscopic examination, especially for distinguishing men with probable optimal fertility potential.

The correlates and alleged biochemical background of the resazurin reduction test in semen

“…Glass et al . (1991 ) and Rahman & Kula (1997) found the result of the resazurin reduction test by semen samples to be significantly correlated with the concentration of motile spermatozoa. Mahmoud et al .…”

“…The resazurin reduction test is used to detect metabolically active spermatozoa. The reaction causes a visible change of colour from blue to pink ( Glass et al ., 1991 ; Fuse et al ., 1993b ; Rahman & Kula, 1997). In the current study we applied a photometric method to assess the decrease of the resazurin blue in the extracted colour in a quantitative way, and we have investigated the biochemical basis of the resazurin reduction reaction.…”

“…This reaction is manifested by a visual change in the colour from blue to a spectrum ranging from dark purple to pink ( Erb & Ehlers, 1950; Glass et al ., 1991 ). The reduction of resazurin by human semen is correlated with the number of motile spermatozoa ( Glass et al ., 1991 ; Rahman & Kula, 1997), and with the concentration of ATP in semen ( Mahmoud et al ., 1994 ). The result of the resazurin test has been found to be influenced by the oxidative stress of spermatozoa and by prostatic function ( Zalata et al ., 1995 ).…”

Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality

“…It has been mostly used for the evaluation of human semen (Mahmoud et al. , 1994; Rahman & Kula, 1997; Zalata et al. , 1998; Reddy Venkata Rami & Bordekar, 1999) but, to our knowledge, only once in veterinary medicine for evaluating ram semen quality (Wang et al.…”

Determination of the diagnostic value of the resazurin reduction assay for evaluating boar semen by receiver operating characteristic analysis

“…The level of resazurin reduction can be measured with spectrophotometry and used as a test to quantitatively measure sperm metabolic activity. The assay has been applied to evaluate semen quality in humans [6][7][8][9], rams [10] and boars [11].…”