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W V Holt

Publications and source records attributed to W V Holt.

61 records · Page 4Linked to original sources
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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↗
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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.

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Choice of operating conditions to minimize sperm subpopulation sampling bias in the assessment of boar semen by computer-assisted semen analysis.

The performance of a computer-assisted semen analysis system was evaluated for use with washed boar spermatozoa. Accuracy was tested using a computer graphics-generated series of spots moving along horizontal, vertical, and diagonal paths, with both straight and sinusoidal trajectories. Observed and expected values agreed to better than +/- 5%, and there was exact agreement in many cases. Reproducibility was tested by making 10 measurements of a single prerecorded sequence of boar spermatozoa. Coefficients of variation were < 3% for all sperm motion parameters tested. Setup conditions affecting the sample statistics of sperm populations were examined. Search radius (10 settings) and minimum track point (10 settings) were varied factorially to evaluate their biasing effects upon population sampling and accuracy. Low search radius (< 12 microns) or high minimum track point values (> 26 frames) precluded measurements of rapidly moving cells and thus led to selection of slow-moving cells. High search radius (> 16 microns) and low minimum track point settings (< 22 frames) led to erroneous tracking and poor data quality. Suitable settings for these setup parameters (search radius = 13 microns; minimum track points = 24) were chosen for use in subsequent fertility trials because they caused the least sampling bias.

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Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials.

Two fertility trials were undertaken to evaluate the relationship between boar semen quality and fertility (conception rate and litter size) after on-farm artificial insemination (AI). Trial 1 included 98 ejaculates from 27 boars, and trial 2 included 72 ejaculates from 26 boars. The semen quality was measured by computer-assisted semen analysis (CASA) using the Hobson Sperm Tracker. Boar semen was diluted in a standard extender (Beltsville Thawing Solution, BTS), dispensed into 75 ml allquots each containing 1.5 x 10(9) spermatozoa and dispatched to farms by overnight mail for use by their normal AI procedures. Randomly selected 75 ml aliquots of semen from each boar were also sent to the institute of Zoology for CASA measurement. Prior to CASA analysis, the spermatozoa were recovered from the BTS using Percoll gradients, resuspended in trisbuffered saline media containing 40 mM Ca++, and incubated at 39 degrees C. Parameters of sperm motion were measured after 0, 2, 4, and 6 hours of incubation. Various multiple regression models based on measured motion parameters could account for up to 24% of the variation in litter size. Using logistic regression, highly significant (P < 0.0001) models explaining conception rate in terms of sperm motion were derived for trial 2 only. The change in sperm velocity during the first 2 hours of incubation and the magnitude of the velocity parameters after 2 hours were identified as the most consistent indicators of fertility. Other attributes of sperm quality, i.e., frequency of spontaneous acrosome reactions (AR) and ARs induced by ionophore A23187 or solubilized pig zona pellucida, were also examined. When the "within-trial" median litter size was used as a way of allocating ejaculates to "high" or "low" litter-size groups, higher litter size was associated with lower frequency of both spontaneous and induced AR. These results demonstrate that fertility information can be derived from the CASA analysis of boar semen provided it is combined with a period of incubation in capacitating conditions.

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