A comparison of changes in the acrosomes of deep-frozen ram and bull spermatozoa.
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Animals↗
Biomedical subjects
P F Watson
Publications and source records attributed to P F Watson.
Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation.
This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) that morphologically distinct subpopulations of spermatozoa exist within fresh boar ejaculates and that the incidence of these subpopulations is correlated with semen quality following cryopreservation. Five ejaculates were collected from each of 15 boars (5 boars from each of 3 breeds). An objective sperm morphology analyzer used Fourier shape descriptors to describe variation in the morphology of 300 spermatozoa per ejaculate before freezing. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and 5 straws (0.5 mL) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells and motility characteristics (with computer-aided sperm analysis), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (fluorescein-labeled peanut agglutinin positive). Consistent between-boar variability was detected for post-thaw sperm motility (P < .01), membrane integrity (P < .01), acrosome integrity (P < .01), curvilinear velocity (P < .01), straight-line velocity (P < .05), beat cross-frequency (P < .05), and amplitude of lateral head displacement (P < .01). Three morphologically distinct subpopulations of spermatozoa, defined by Fourier descriptors, were detected. The proportion of these subpopulations within the fresh ejaculate correlated with semen quality assessments made following cryopreservation. These findings support the hypothesis that consistent interindividual variation in sperm freezability is genetically determined and may relate to processes that occur during spermatogenesis. Subsequent characterization of these genetic differences between "good" and "poor" freezers may ultimately identify biophysical components of the spermatozoa that are essential for successful cryopreservation.
Acrosome↗
A custom-built controlled-rate freezer for small sample cryopreservation studies.
A custom-built controlled-rate freezing machine is described. It operates by automatically raising and lowering the sample carrier in a static column of liquid nitrogen vapour. It is controlled by a computer-assisted thermocouple feedback system that operates a stepper motor driving the sample carrier. The cooling protocol is divided into three phases: cooling from +5 to -5 C, initiation of ice nucleation, and cooling from -5 to -80 C. Experiments are described to validate the device over a range of different cooling rates. A freezing protocol is established to cool samples in plastic straws over a range of rates up to 80 C/min, with rapid and consistent absorption of the latent heat.
Animals↗
A comparison of critical osmolality and hydraulic conductivity and its activation energy in fowl and bull spermatozoa.
Measurements were made of critical osmolality, the osmolality at which 50% of the cells are lysed, and of the permeation time, the time taken to lyse 50% of the cells in an osmotic solution lower than the critical osmolality, for fowl and bull spermatozoa. Cell lysis was determined by means of fluorescent viability stains (carboxyfluorescein diacetate and propidium iodide) using a flow cytometer. The advantages and pitfalls of this approach are addressed. The values obtained have been used to compute the water permeability, or hydraulic conductivity, of the plasma membrane and its activation energy for each species. Fowl spermatozoa were found to have a lower critical osmolality (17 mOsm) than bull spermatozoa (36 mOsm), and this is discussed in relation to the differences in cell shape and size. The hydraulic conductivities of fowl and bull spermatozoa were 2.1 and 10.8 microns x atmosphere x minute, respectively, and the respective activation energies were 4.4 and 3.0 kcal/mol. The relevance of these findings to cryopreservation of spermatozoa is considered.
Animals↗