الفهرس 
List of abbreviations
Semen cryopreservationOnly 14 pages are availabe for public view
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Abstract
This study aimed to : evaluate the damaging effects induced at each processing step of freezing semen on functional (motility, livability and abnormality) and ultrastructural characteristics using scanning and transmission electron microscope of bull semen. Different processing steps are involved (dilution of semen at 37°C with Tris-based diluents containing egg yolk and glycerol. This is followed by cooling to 4°C and an equilibration period. Rapid freezing is then performed to avoid crystallization. We also compared different methods (Toluidine blue staining, Acridine orange fluorescence staining and Sperm chromatin Structure Assay) of assessment of sperm chromatin damage.The obtained results can be summarized as follows Raw and freshly diluted semen samples maintained at 37°C exhibited similar motility and viability. After initiation of cooling and during the equilibration period, significant reduction in sperm motility (P<0.05) was associated with a significant decrease in sperm viability and increase in the percentage of sperm abnormalities (mainly coiled and bent tails). Examination of sperm cells using TEM revealed that the PM, particularly surrounding the sperm head, was remarkably affected, and this effect varied at different processing steps. Stages of semen cooling and freezing did not affect the number of spermatozoa with slight swelling in PM compared to diluted semen at 37°C (P>0.1). Marked swelling significantly increased after 4h equilibration (P = 0.005) and further after freezing (P<0.0001). Freezing, but not cooling, also resulted in a complete loss of PM in a significant number of spermatozoa (P=0.032). Also using a scanning electron microscope as shown damage in Plasma membrane (PM) after the freeze but cracking deeper damage, when compared with other stages before freezing. Freezing significantly increased typical acrosome reaction compared to diluted semen (37°C) (P=0.002) and also increased atypical AR (P=0.046) and tended to increase the probability of complete loss of acrosome (P=0.91). Cooling and equilibration did not induce significant changes in the acrosomal structure.Using scanning electron microscopes was shown damage in acrosome after the freezing when compared with other stages before freezing. In fresh semen samples, normal sperm head containing normal nucleus with homogenous condensed chromatin surrounded by intact plasma membrane and acrosome were noted. In contrast, an increased percentage of spermatozoa showing signs of nuclear damage and loss of chromatin integrity could be seen in frozen-thawed samples. By TEM examination of sperm mid-piece, we investigated the morphology of the mitochondria. In fresh semen, mitochondria appeared normal with a membrane space of variable width and clearly visible cristae. After cryopreservation, mitochondrial damage was observed in the form of increased number of mitochondria with distorted cristae. Damaged mitochondria appeared vacuolated with narrowed membrane space By TEM examination of sperm end-piece, we investigated the morphology of the axoneme. In fresh semen, axoneme appeared normal with intact plasma membrane, normal axoneme, microtubules principle (9x2) and microtubules central’s (2). After cryopreservation, axoneme damage was observed in the form of increased number of Microtubules principle with distorted, 5x2. Also using scanning electron microscope was shown increased coiled tails after the freezing when compared with other stages before freezing. Techniques of Toluidine blue and acridine orange had highly significant effect (P≤0.001) in the estimation of the percentage of chromatin damage during different stages, while higher values (4.5 % for TB and 5.3 % for AO) were showed during freezing and thawing stage and lower values (1.5 % for TB and 1.16 % for AO) were showed in row semen.On the other hand, by the using of Flow cytometery the percentages of chromatin damage were ranged from 2.79 % after dilution at 37 °C to 8.42% during freezing and thawing stage.In conclusion, using standard freezing protocols with glycerol and egg-yolk protection in tris-based extenders, the most pronounced damage induced by cryopreservation is observed in the plasma membrane structure. Acrosomal, mitochondrial and chromatin damage are also evident but appear to be within acceptable limits, but can be further reduced by targeted supplements. We highlighted that reducing damage in sperm plasma membrane, mitochondria and DNA should be assessed and targeted at during equilibration when most of the damage is initiated for improving freezing protocols of bull semen used for AI. This should be also applied on selection of sires using TEM and SCSA as described above. Briefly, the present study indicated marked difference in impairment of semen quality and chromatin damage during different steps of Friesian bull frozen spermatozoa, in particular, from equilibration to freezing/thawing.