Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

Abstract

Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

Paper-Based Resazurin Assay of Inhibitor-Treated Porcine Sperm

“…Figure 3A suggests that 3-BP treatment induces a decrease in motility, as previously reported ( p < 0.025) [33]. RBCI values for treated and untreated samples, as shown in Figure 3B, indicate that the average RBCI values for 3-NPA treated and 3-BP treated samples were significantly lower than that of the untreated samples ( p < 0.025).…”

“…It was gently mixed before and after adding inhibitor agents, which were allowed to incubate with our sample for 30–90 min. We used iSperm (Aidmics Biotechnology, Taipei, Taiwan) to analyze inhibitor-treated samples as per our previous report [33]. Sperm concentration in the semen was from 500 to 1100 million sperm cells/mL.…”

“…Previous experiments were completed using photometric devices [15,16,18,20,21,22]. To provide precise, robust, portable, and inexpensive redox-based assay systems, paper-based assays have been developed for mammalian sperm using MTT [31,32,33,34]. We found a correlation between colorimetric results from a paper-based MTT assay and sperm motility as determined by inhibitor treatment, and suggested that the redox mechanism was related to flagellum beating [31,32].…”

Validation of a portable device (iSperm®) for the assessment of stallion sperm motility and concentration

“…The iSperm ® device was introduced as a practical and relatively inexpensive device for measuring motility and concentration of spermatozoa in semen from a variety of domestic species including poultry, pigs, ruminants and dogs (Matsuura, Huang, Chen, Chen, & Cheng, ; up iSperm App). In recent years, algorithms have been optimized for measuring equine semen parameters.…”

The Roles of NO and H2S in Sperm Biology: Recent Advances and New Perspectives

“…Moreover, Mustafa et al [64], upon the observation of H 2 S interaction with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), suggested H 2 S to be antagonistic to NO, since H 2 S tends to increase cysteine reactivity rather than decrease it, as in the case of NO. This finding may have interesting implications in sperm biology, since GAPDH is a glycolytic enzyme involved in sperm motility [65,66]. In addition, a sperm-specific isoform (GAPDS) with constitutional differences and more specific function is found within sperm cells [67].…”