Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen

Abstract

Cryopreservation of semen imposes deleterious effects on spermatozoa, either killing a certain proportion of cells or causing subtle damages on sperm function in the surviving population, changes not easily revealed by conventional assays. We have tested three functional assessment techniques in frozen-thawed ram semen from six adult rams, cryopreserved following eight different protocols (four extenders, and glycerol being added at two temperatures). Semen samples were thawed and the following analyses were carried out: motility (CASA), membrane integrity (Hoescht 33258 and fluorometry), chromatin status (chromatin stability test and fluorescence-assisted cell sorting, FACS) and mitochondrial activity (JC-1 and FACS). Fluorometry outcome did not correlate with the other parameters and showed large variation, albeit discriminating among cryopreservation techniques (P<0.01). Mitochondrial activity correlated, but with low values, with total and progressive motility. However, good sperm motility and high velocity values were associated to high mitochondrial membrane potential. The chromatin stability assay was also successfully carried out, and had a good relationship with male factor (%COMP α_t and SD α_t parameters). In conclusion, fluorometric assessment of membrane integrity albeit rendering poor results, merits improvement, being a low-cost and handy technique, especially for work in the field. On the other hand, both assessments of chromatin stability and mitochondrial status (JC-1 staining), combined with FACS, are reliable techniques that can be used for the functional assessment of frozen-thawed ram semen.

Introduction

Functional sperm analyses have been gaining importance during the last decades, since conventional techniques have not demonstrated to be able to accurately and repeatedly estimate the fertility of a semen sample (Correa et al., 1997). Thus, the development of techniques that pursue to evaluate the functional status of sperm organelles (acrosome, mitochondria) or the integrity of many cellular components (membranes, chromatin) (Graham, 2001), allows a different approach to the problem. In this work we have focused on membrane integrity, chromatin integrity and mitochondrial status.

Membrane integrity is a fundamental requisite for sperm viability and the success of fertilization. There are many techniques based in the use of permeating and non-permeating dyes, so membrane damaged cells can be detected. The combination of fluorescent probes and fluorescence microscopy or flow cytometry has proved to be objective and accurate Harrison and Vickers, 1990, Garner et al., 1994, but the methods are slow (microscopy) or costly and unpractical in the field (flow cytometry). Instead, the use of an automated fluorometer is a low-cost and rapid approach that can be readily used on a routine basis Gravance et al., 2000, Alm et al., 2001, Januskauskas et al., 2001.

Mitochondrial status plays an important role because of its relationship with the energetic status of the cell and motility, and has been related to fertility Casey et al., 1993, Kasai et al., 2002. In this case, the fluorescent dye JC-1 has successfully been used to estimate mitochondrial membrane potential in sperm, both by fluorescence microscopy and flow cytometry Garner et al., 1997, Papaioannou et al., 1997, Thomas et al., 1998, Troiano et al., 1998, Gravance et al., 2000.

Another important factor for sperm functionality is the integrity of the nuclear chromatin. This can be evaluated using a test where DNA denaturation in situ is performed (Evenson et al., 1980), which is a well-established technique that has proved its utility in many species Dobrinski et al., 1994, Evenson et al., 1994, Sivashanmugam and Rajalakshmi, 1997, Muratori et al., 2000, Spano et al., 2000, Blottner et al., 2001. Defects in the structure or packaging of the chromatin can severely impair fertilization or embryo development Spano et al., 2000, De Jonge, 2002. Thus the test has been used for fertility estimation, as well as detection of problems during spermatogenesis Dobrinski et al., 1994, Evenson et al., 1994, Januskauskas et al., 2001, Blottner et al., 2001.

Information about the use of these techniques in ram semen is scarce. We have analyzed frozen-thawed ram semen samples, that were cryopreserved using eight different protocols. Our objective was to test fluorometry, JC-1, and chromatin stability assay with frozen-thawed ram semen, not only to assess their usability with this species, but also to look for differences between the cryopreservation protocols.