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

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.