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Title: Studies on the spermatozoa of the ram, with special reference to the effects of deep freezing
Author: Alsaadi, Hussain A. K.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1977
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This thesis describes studies on the spermatozoa of the ram carried out with a view to determining the effects of deep freezing which lead to poor fertility following artificial insemination. The electro-ejaculator was employed for semen collection over 20 months and 80 samples out of 114 collections were processed. Motility of spermatozoa was assessed by scoring mesa activity in raw semen samples and by estimation of the percentage of motile spermatozoa in raw and processed samples. Percentages of live spermatozoa and their general morphology were studied in eosin-nigrosin stained smears and aoroaomal defects in eosin fast green FCF stained smears. Spermatozoal morphology was also studied with the electron microscope. Generally the various parameters of the raw semen were within or close to the standard ranges and they varied with season, beat semen being collected in the autumn. When semen was frozen by a standard technique there was a continuous reduction in the spermatozoal viability, especially motility, associated with an increase in morphological deterioration of the spermatozoa, especially of their acrosomes, following each stage. At the same time variation in spermatozoal viability and morphology existed between different samples. Various modifications of the freezing technique were tried. On inclusion of 2-Wt glycerol or dimethyl sulphoxide (DM80), or their combinations at different levels in ane yolk and lactose diluent the results respectively showed that 4% glycerol, 3% DMSO andozoa equilibration times (0.5-24.0 hours) dilution rates of (1:1-11:10), thawing media (sodium chloride or citrate with or without lactose in solution at 37°C or frozen pellet at -196°C) and thawing temperature (000-1000C) were studied using both 4% glycerol or 3% DMSO as the cryoprotective in the diluents. The results showed that equilibration of 3.0 hours with glycerol, and of 1.9 hours with DPSO, and dilution rates of 1:4 and thawing directly in a dry test tube at 37°C-100°C, irrespective of the cryoproteotive, were the optima. In addition, various methods of dilution (dropping or direct at 4°C or 20°C or their combination) and different egg yolk levels (25% or 50%) with or without sodium citrate (3%) were used. The results indicated that equilibration at 2000, irrespective of method of dilution led to a high death rate of ram spermatozoa but provided surviving spermatozoa some resistance against cold shock during freezing. Direct addition of the diluent irrespective of the equilibration temperature was satisfactory, but the addition of the diluent by dropping for 0.5 hour at 30t followed by 1.0 hour equilibration at 4°C was superior. Increasing the egg yolk level from 25b to 5C, with or without sodium citrate in 4% glycerol containing diluent was harmful which might be the result of binding up of glycerol. Prompt dilution of the raw semen after collection and the avoidance of temperature fluctuation between stages of freezing and when sampling for thawing were tried and the results showed an enhanced resistance to cold shock and an improvement in spermatozoal motility when thawed after 24 hours storage at -196°C. However, longer storage was still deleterious, and the percentage of motile spermatozoa fell to 4C. The post-thawing life span and morphological changes of the ram spermatozoa were evaluated following thawing at 0°C-100°C and incubation, at 39°C for 0, 3.0 and 6.0 hours. The results showed that the life span of the frozen-thawed ram spermatozoa was short (around 3 hours) and their acrosomal defects increased progressively as the incubation time increased. The fertilizing efficiency of frozen semen stored for 42-46 days was tried on 28 ewes, but the results showed that the low post-thawing motility and short survival time which pertained were not adequate to produce pregnancy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available