A Comparison of Critical Osmolality and Hydraulic Conductivity and Its Activation Energy in Fowl and Bull Spermatozoa

One effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post‐thaw is lost more rapidly at later times than in fresh cells. We hypothesized two modes of sublethal cryodamage: one is peroxidation‐related involving plasma membrane damage due to lipid peroxidation; the other is membrane stress‐related involving membrane embrittlement during phase transitions occurring during freeze‐thaw. If the peroxidation‐related mode contributed substantially to sublethal cryodamage, the hypothesis predicts that lipid peroxidation inhibitors should reduce this damage. To test this prediction, we examined the effect of the lipid peroxidation inhibitors, hypotaurine, bovine serum albumin (BSA), and α‐tocopheroi (Vit. E) on the time to loss of motility (TLM), taken as a measure of cell viability over time, for sperm samples cryopreserved in glycerol plus egg yolk medium. These agents had no effect on TLM of these samples, indicating that this mode contributes little to sublethal cryodamage. If the membrane stress‐related mode contributed, the hypothesis predicts rapid recovery of motility in the presence of egg yolk plus glycerol, but slow recovery in the presence of glycerol alone. It also predicts that an appropriate polyol may be both necessary and sufficient for cryopreservation. In the presence of egg yolk plus glycerol, motility recovery was complete within 5 minutes, but the percent motile cells then decreased linearly with time. With glycerol alone in the range 3–12%, at 5 minutes post‐thaw the percent motile cells was 5–10%, but by 40 minutes post‐thaw had risen to 60–80%, approaching that in the fresh sample, and was maintained up to 4 hours. In the absence of glycerol, the percentage of motile cells post‐thaw was nil and remained nil up to 4 hours. The polyols, erythritol, ribitol, and sorbitol had similar effects to that of glycerol, but the recovery of motility was not as complete. These results indicate that the membrane stress‐related mode contributes substantially to sublethal cryodamage. They also indicate that glycerol and other polyols can function alone as cryoprotectants, but that recovery of motility is slow in these systems.

Evidence that Membrane Stress Contributes More than Lipid Peroxidation to Sublethal Cryodamage in Cryopreserved Human Sperm: Glycerol and Other Polyols as Sole Cryoprotectant

ALVAREZ,

STOREY

1993

Mechanism of Superoxide Dismutase Loss from Human Sperm Cells during Cryopreservation

LASSO,

NOILES,

ALVAREZ

et al. 1994

113

Earlier studies on human sperm cryodamage have shown that plasma membrane stress is the primary process and that phospholipid peroxidation in cryopreserved samples is not inhibited by addition of antioxidants. One consistent effect of cryopreservation is loss of enzymatic activity of the peroxidation defense enzyme, superoxide dismutase (SOD). To clarify this aspect of the freeze‐thaw process and to develop a more complete resolution of the reactions leading to cryodamage, we sought to identify which of the two most probable mechanisms, loss of enzyme protein from the cells or denaturation of the protein, operates. If the first operates, cellular enzymatic activity and enzyme protein as identified by immunocytochemistry should give a linear correlation. If the second operates, there should be no correlation. In this study, five individual samples were analyzed before and after cryopreservation for immunoreactive Cu/Zn SOD and cell intactness by flow cytometry, for SOD enzymatic activity by a highly sensitive fluorimetric method, and for motility characteristics by Hamilton‐Thorn motility analyzer. Fresh samples were obtained by the “swim‐up” method and had >95% intact cells with >78% motile cells. After freeze‐thaw, about half the cells were intact. SOD enzymatic activity was determined on Triton X‐100 cell extracts, a method that removes all enzymatic activity from the cell structure, and compared with immunoreactive SOD in the cells as determined by indirect immunofluorescence mean intensities. Residual immunofluorescence was observed in the cells after Triton X‐100 treatment; if this was taken into account, a close linear correlation between SOD enzyme activity and SOD immunoreactivity was obtained (r = 0.90; P = 0.00014). There was no correlation between SOD enzyme activity ratios for crypreserved and fresh cells and fraction of intact cells after freeze‐thaw. We conclude that loss of SOD protein from the subset of cells undergoing acute membrane damage is the most probable primary mechanism of SOD enzymatic activity loss from the sample and that resistance to cryodamage and SOD activity in any given cell are quite independent of one another.

Dual DNA Staining Assessment of Bovine Sperm Viability Using SYBR‐14 and Propidium Iodide

GARNER,

JOHNSON,

YUE

et al. 1994

193

A new membrane‐permeant DNA stain, SYBR‐14, was used in combination with propidium iodide (PI) to estimate the proportion of living sperm in bovine semen. The SYBR‐14 stained living sperm while PI only stained degenerate cells that had lost their membrane integrity. Staining with SYBR‐14 resulted in the nuclei of living sperm fluorescing bright green. Aliquots containing nearly all living bovine sperm were prepared using glass wool/Sephadex filtration to remove dead and damaged cells. A portion of this filtered sample was killed by unprotected freeze‐thawing and used to provide mixed aliquots containing known ratios of living and dead sperm. Flow cytometry was used to assess the green and red fluorescence of these mixtures. The percentages of living sperm, as determined by the log of green fluorescence, were 85.1, 68.8, 39.8, 20.7, and 1.4 for ratios of 100:0,75:25,50:50,25:75, and 0:100 of the filtered, killed mixtures. Also, bovine semen was diluted 1:60 in HEPES‐0.1% bovine serum albumin and incubated for 0,3,6, and 24 hours at 36°C to assess changes in cell viability. As ceN death occurred during this incubation period, a relatively rapid transition of staining from green to red occurred as sperm died. Three replicates of cryopreserved sperm from six bulls were also examined using SYBR‐14 and PI to assess the proportion of living and dead cells. Flow cytometric analyses of these samples, which had been processed and stored in homogenized milk, indicated that this stain combination was useful in assessing the quality of cryopreserved sperm. The combination of SYBR‐14 and PI was determined to be an effective tool for assessing the viability of fresh or cryopreserved sperm.