Sperm recovery and function testing. Results for the three experimental series with pooled spermatozoa are presented in Table I
. These are summarized below in two parts, those from linear cooling rate and passive vapour freezing, and those where the extracellular conditions were specifically controlled.
Linear cooling rates and passive vapour freezing (Methods 1–5). In experiment #1, samples cooled in the Planer freezer using method (3), at a linear rate without manual ice nucleation showed a significantly lower recovery rate (recovery and viability) than samples cooled linearly with ice nucleation (method 2) and those cooled linearly in the Asymptote SF100 freezer with manual nucleation (method 4) and without manual nucleation (method 5). In experiment #3, where two different rates of linear cooling were used (10°C/min and 18°C/min) using method (2) there was no significant difference between the two rates. Passive vapour phase cooling (method 1) resulted in similar recovery rates to programmed linear cooling (methods 2, 4 and 5).
Control of extracellular freezing (Methods 6.i, 6.ii, 6.iii). The highest recovery of grade A motility was seen in all three experiments when cooled using the `controlled concentration' method described above (method 6.i). A modification of this protocol, giving an even greater initial rise in solute concentration, was attempted in experiment #3, but gave rise to slightly lower recovery rates.
Those samples cooled such that the ice fraction increased at a linear rate (method 6.ii) had the poorest recovery in terms of motility. In addition, this protocol appeared to have a significant effect on membrane function with hypo-osmotic swelling apparent in all samples immediately on thawing (100% in experiment #1), and a significant decrease in the ARIC score in all three experiments.
Linear heat extraction (method 6.iii), which was carried out only in experiment #2, gave rise to a significantly lower recovery rate in terms of motility, viability and function, compared with passive cooling, and `controlled concentration'. It should also be noted that the sample used in experiment #2 appeared to be more susceptible to cryodamage than the samples used in experiments #1 and #3.
Experimental series #4—non-pooled sperm. Spermatozoa from three patients were frozen by both the controlled concentration (method 6.1) and conventional linear cooling (method 4) and the results are presented in Table II. These three samples varied in their sensitivity to freezing injury when frozen by conventional linear cooling. When frozen by the controlled concentration method these samples retained their relative ranking, however motility compared with linear cooling was increased by a factor of at least 50%.
Temperature histories within the straws. The concentration–time histories of the samples during freezing by the various methods could be derived from the measured temperature changes. It is of particular importance, and the purpose of the work described here, to control the rate at which the extracellular solute concentration is changing. This rate of change is illustrated in Figure 1, for three of the most distinct cases: `controlled concentration' (method 6.i), linear ice fraction (method 6.ii) and the standard linear cooling rate (method 4). The proportion of time for which this rate of change is decreasing is critical, as shown in Figure 2 where the percentage of time that the rate of change is decreasing was calculated and plotted against post-thaw recovery of motility in spermatozoa for experiments #1 and #3 (experiment #2 was excluded as there were too few data points). There was a clear direct correlation between these parameters. (Data from passive vapour freezing and from treatments where no manual nucleation took place was also excluded because without manual nucleation there would be significant unknown straw-to-straw variation.)
Electron microscopy. Cross-fracture of the straws followed by deep etching to remove ice revealed the structure of the freeze-concentrated glycerol (Figure 3a, b) which had a uniform appearance across the straw. The revealed ice crystal structure showed a similar structure and spacing of ice crystals in all materials frozen by the different methods in which ice was manually nucleated at –5°C.
At higher magnification the freeze-etched samples revealed that the sperm cells had migrated into the freeze-concentrated material during solidification: spermatozoa were not entrapped within ice crystals. However some sperm tails were observed to extend away from the freeze-concentrated material, suggesting that these structures were associated with or entrapped in ice (Figure 3c
). Occasionally spermatozoa were observed with the sperm head entrapped in one portion of freeze-concentrated matrix with the sperm tail tethered in a distinct zone with the intervening tail bridging a void that would have contained an ice crystal (Figure 3d). Some spermatozoa were associated with the interface between the freeze-concentrated material and ice crystals (Figure 3e), but generally few spermatozoa were apparent, but these spermatozoa did not appear to be osmotically shrunken. Occasionally very distorted spermatozoa were observed (Figure 3f); these were apparently entrapped between ice crystals rather than osmotically dehydrated.
Light microscopy of freeze-substituted sections showed that spermatozoa were entrapped within the freeze-concentrated material (Figure 4a, b). Occasional spermatozoa were observed to bridge across two freeze-concentrated zones. At this magnification the freeze-concentrated matrix was observed to be relatively homogeneous in appearance following both linear cooling and linear ice fraction solidification (methods 4 and 6.ii). However following `controlled concentration' (method 6.i) areas of granularity were evident (Figure 4b), which may be interpreted as substituted ice crystals within the freeze-concentrated matrix.
When observed by electron microscopy (Figure 5
) the freeze-concentrated matrix was electron dense, presumably due to the protein components of the egg yolk included in the cryoprotectant. All samples were observed to contain substituted ice crystals in the freeze-concentrated matrix. Frozen spermatozoa in the freeze-concentrated matrix were surprisingly similar to unfrozen controls. There was no evidence of osmotic shrinkage or of the presence of ice voids within the heads of the spermatozoa for any of the freezing methods examined. Occasionally some shrinkage was observed in distal sections. Many sections of sperm head and tails were surrounded by a zone, less than 0.1 µm wide, of low electron density material.
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