Tris buffer improves fluorescence yield of ram spermatozoa when evaluating membrane integrity

Abstract

This study was designed to evaluate the effect of various buffers on the fluorescence signal intensity of two fluorochromes (IP and CFDA) when used to assess the membrane integrity of ram sperm. Second ejaculates (18) from nine adult males were collected using an artificial vagina and diluted in either MOPS, TRIS, TES, HEPES, citrate, or phosphate-based extenders. Semen samples were stored at 15°C and the membrane integrity was assessed within the first 24 h of storage. Mean fluorescence intensity (FI) of PI- and CDFA-labeled sperm heads and fluorescence background noise (FBN) were determined quantitatively using Image J software. Fluorescence contrast (FC) was expressed as the difference between FI and FBN. Significantly, higher FI and FC were recorded when TRIS diluent was used, rather than the other diluents, both in the propidium- and fluorescein-labeled cells. The citrate and phosphate-based extenders showed intermediate results of FC between those of TRIS and zwitterionic (MOPS, TES and HEPES) groups for the PI-labeled sperm. However, in the CFDA-labeled sperm, the lower values of FC were obtained in the citrate and phosphate groups due to increased levels of FBN. For the membrane-damaged sperm, fluorescent labeling was limited to the sperm heads when TRIS-buffer was used, whereas in the other groups, the sperm tail was also frequently observed. It was concluded that TRIS buffer solution markedly increases the fluorescence yield of IP/CFDA-labeled sperm cells in the ram and that this should be considered when evaluating their membrane integrity.

A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method

“…The criterion for inclusion in the study was 70% MS because only these samples are typically used in the industry. Within the first 24 h of storage, the semen samples were carefully mixed and diluted to 100 × 10 6 spermatozoa ml −1 with a TRIS-based solution12 immediately before processing for sperm morphometric analysis. In boars, semen was diluted in Bio’dil® (Genes Diffusion, Douai, France) to 50 × 10 6 spermatozoa ml −1 , kept in sterile plastic bottles and stored in a refrigerator at 15°C.…”

Use of Relief Contrast®Objective to Improve Sperm Morphometric Analysis by Isas®Casa System in the Ram

“…Within the first 24 h of storage, the semen samples were carefully mixed and diluted to 200 × 10 6 spermatozoa/ml with a TRIS‐based solution (Yániz et al. ) immediately before processing for sperm morphometry analysis.…”

Effect of diluent composition on the dynamics of sperm DNA fragmentation and other sperm quality parameters in ram during incubation at 37°C

“…In some diluents, such as CITRATE, the pH decrease during incubation was not accompanied by an increase in SDF, although the pH drop was less pronounced than that observed in the MILK and INRA extenders. Some buffers are not physiologically inert and may have different effects on cells (Yániz et al, 2012). For example, buffers can act as metal ligands, affecting several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction (Spear and Aust, 1998).…”