1972 Reproduction  
The results from the insemination of frozen-thawed bull semen approach or equal those expected from natural mating, and contrast with the poor results obtained using frozen-thawed ram semen (Emmens & Robinson, 1962) . Healey (1969) , describing the ultrastructural changes in spermatozoa resulting from freezing, noted differences between species in the degree of acrosomal deterioration. This report summarizes the results of experiments on the cooling and deep\ x=req-\ freezing of ram and bull
more » ... rmatozoa. The degree of change in the acrosomes was estimated by means of light microscopy after the specimens had been stained with Giemsa, using the following scoring system: 0, normal acrosome; 1 and 2, stages of acrosomal damage; 3, acrosome entirely lost. A typical spermatozoon in each of the categories is shown in Text- fig. 1 . Ejaculates from fifteen bulls were examined in one experiment and from twelve rams in a second experiment. Semen was diluted tenfold at 30\s=deg\C and cooled to 5\ s=deg\ C at a constant rate of 0\ m=. \ 27\ s=deg\ C/min. Further diluent, containing glycerol, was added at 5\ s=deg\ C to bring the dilution to twentyfold, and the semen was frozen in ampoules containing 1-ml portions after equilibration at 5\ s=deg\ C for 1 hr. The freezing rate was approximately 3\ s=deg\ C/min to \ m=-\ 40\ s=deg\ C, followed by rapid cooling to \m=-\196\s=deg\C. After storage for less than 1 hr, frozen samples were thawed by immersion of the ampoules in a water bath at 37\s=deg\C. The final composition of the diluent was 205 niM-glucose, 17 mM-fructose, 49 mM-NaCl, 5 mM-KCl, 10 mM-Na2HP04, 10 mM-NaH2P04 6% egg yolk, v/v, and 7-5% glycerol, v/v. Before the addition of glycerol to the second-stage diluent, both diluents were centrifuged for 10 min at 1000 g to remove the larger globules of egg yolk. Smears were made from samples of diluted semen at 30°C , at 5°C after equilibration with glycerol, and after freezing and thawing. The smears were dried in warm air, fixed in neutral formol-saline (5% formaldehyde) for 15 min, rinsed in tap-water, and stained in the following solution: 3-0 ml Giemsa (Gurr R66), 2-0 ml Sorensen's buffer (pH 7-0) and 45-0 ml distilled water. All slides within each experiment were examined in random order and were so labelled that the observer did not know from which treatment the sample came. The acrosomes ofthe first twenty spermatozoa observed in a traverse of the smear were examined, and the mean score for these spermatozoa then became the unit of data for analysis of variance.
doi:10.1530/jrf.0.0280099 pmid:5008009 fatcat:vvwv6pmjdje5bdfh54smsy4qne