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For dog breeders as well as for veterinarians the aim of a semen evaluation is to give a prognosis about the fertilizing capacity of a semen sample. Conventional semen analysis includes determination of volume, color, sperm concentration, progressive sperm motility, sperm membrane integrity and sperm morphology. Besides, several additional parameters have been evaluated like the hypo-osmotic swelling test (HOS test). The HOS test is an easily applicable test to assess the functional integrity of the plasma membrane of sperm cells.
The overall objectives of this thesis were to analyse relationships between HOS test results and conventional semen parameters before freezing and after thawing and to determine the diagnostic value of the HOS test as a standard procedure for canine semen evaluation.
In preparation for the clinical trial, a systematic literature review was conducted in order to evaluate the quality of published literature concerning the HOS test. Using two databases, 354 articles were found. Out of these, 38 articles were eligible for further analyses according to specific quality parameters such as randomization, blinding, inter- and intraobserver agreement and sample size. Also test characteristics, such as study design, population, semen sampling and implementation concerning the HOS test were investigated. A citation map was developed to identify the origin of the quotation of the HOS test. Although there were numerous articles available, the diagnostic value of the HOS test remained unclear. Until then, neither a recognized test implementation nor reliable reference values have been defined. Most of the trials evaluated showed serious methodological flaws and therefore did not permit drawing reliable conclusions. According to our results, approximately half of the studies (n = 20) included a sample size of five or less animals. None of the studies examined the inter- or intraobserver agreement for the HOS test. Overall, the results of the study indicated the need for standardization of test implementations as well as for reliable reference values. Furthermore, a possible correlation between the HOS test results and the fertilizing capacity will have to be clarified to determine the diagnostic value of the HOS test.
The objectives of the second study were 1) to examine relationships between HOS test results and conventional semen parameters before freezing and after thawing and 2) to evaluate the prognostic value of the HOS test results on the post-thaw quality of canine semen. Semen of 35 dogs was collected and analysed before freezing and after thawing following a 7-day freeze-thaw interval. Conventional semen variables such as sperm cell motility, membrane integrity and morphology were evaluated and also the HOS test was conducted. For the prediction of individual cryopreservation capacity, semen was classified into good and poor quality according to certain semen characteristics. Results from the assessment of fresh semen parameters of good and poor semen quality were statistically compared. Based on these results, it is not possible to predict the quality of frozen-thawed dog semen using the HOS test.
The objective of the third experiment was to examine time-dependent changes of motility after thawing cryopreserved canine semen. Semen motility is expressed as the percentage of total motile or progressive motile sperm cells. It is usually estimated by visual inspection using a light contrast microscope at X 100 at 37°C immediately after semen collection or immediately after thawing frozen semen and should be one of the first steps of a semen analysis. Standard operating procedures, however, have never been established for this test. For this experiment, semen of 35 dogs was collected and volume, concentration, progressive motility, morphology, membrane integrity and HOS test were evaluated. For cryopreservation CaniPRO® Freeze A&B was used. Semen was thawed and diluted using CaniPRO® Culture Medium. After thawing, semen was evaluated as before. In addition, every sample was tested for progressive motile sperm cells 0, 5, 20 and 60 minutes after thawing. Overall, the study revealed significant positive correlations between progressive motility and conventional semen parameters, which were highest five minutes after thawing. To obtain comparable results for motility assessment of cryopreserved canine semen, the development of standard operating procedures was recommended.
The presented three studies indicate the need for standardization of test implementations as well as for reliable reference values to warrant the comparability of information about semen quality. Although significant correlations were found between the HOS test results and conventional semen parameters, the HOS test is not suitable to give a prognosis about the quality of frozen-thawed canine semen.