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 S.D.(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

Antioxidant effects of lycopene on bovine sperm survival and oxidative profile following cryopreservation

“…Sperm cryopreservation has been repeatedly associated with axonemal alterations, leading to disruptions to sperm motion behaviour and mitochondrial membrane potential, ATP depletion, premature acrosome reaction and increased morphological alterations (de Lamirande and Gagnon 1992;Ball 2008). Important associations have been reported to exist between sperm motility, stability of the membranous structures and mitochondrial metabolism (Martinez-Pastor et al 2004). At the same time, we must keep in mind that motility is an essential prerequisite for the male gamete to penetrate the cumulus cells and fuse with the ovum (de Lamirande and Gagnon 1992).…”

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…83 The mitochondrial stain 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Figure 5) that exists as a monomer at low concentration yielding green does permit a distinction to be made between spermatozoa with poorly and highly functional mitochondria. 84 Martinez-Pastor et al 85 observed some relationship between JC-1 staining and motility, although correlation with motility is regulated by many factors. In highly functional mitochondria, the concentration of JC-1 inside the mitochondria increases and the stain forms aggregates that fluoresce orange.…”

Energy Management of Mature Mammalian Spermatozoa

“…There is not a universal agreement regarding the importance of mitochondria-based energy obtainment. In this way, an optimal mitochondrial function has been related not only with sperm motility in bull [18], horse [20], ram [34] and mouse [39] but also with fertilization ability in human (30). However, gene knock-out of the glycolytic enzyme glyceraldehyde-phosphate dehydrogenase (GAPDH) in transgenic mice caused the appearance of non-motile sperm and a significant reduction of the ATP content (10% of the total) despite having no deficiency in oxygen consumption [38].…”