Effekter av spädningsvätska på motilitet och fertilitet hos hingstsperma : en jämförelse av subjektiv motilitetsbedömning och QualispermTM

Abstract: At SLU, Divison of Reproduction, Dept of Clinical Science, there is an ongoing project on the development and evaluation of new methods for the assessment of stallion semen quality. This EEF-study, which is a part of that larger project, has focused on the effects on motility and fertility of stallion semen of two different extenders and selection. Both an objective method and a subjective method for analysis have been used and compared in the study. The increase in the use of artificial insemination with cooled semen in Swedish horse breeding over the last decade is thought to play a part in declining foaling rates (Kareskoski et al, 2006, Kuisma et al, 2006). This calls for better evaluation methods of semen quality since cooling and transportation put higher demands on semen quality in order to maintain fertility (Aurich, 2005). Visual assessment by light microscope is a subjective method of sperm motility evaluation and the most common method used in the field. The correlation between fertility and the subjective motility results are not conclusive which has lead to the development of objective methods for semen analysis. The method evaluated in this project is QualispermTM 2008 (Biophos AG, Switzerland) which is a development of CASA (Computer Assisted Semen Analysis). QualispermTM consists of a computer program and a phase light microscope. The images of thousands of spermatozoa are captured via the camera attached to the microscope. These images are digitalized and analyzed for motility, concentration and speed of movment by the computer. In comparison with other CASA-systems QualispermTM analyses a greater number of spermatozoa, is easier to use and cheaper, which make it more suitable for field-practice. The aim of spermatozoal selection is to increase fertility by sifting out spermatozoa that will keep their fertility potential for a longer time. The selection is carried out through "Single Layer Centrifugation" where the spermatozoa travel through a colloid (Morrell et al, 2008). Centrifugation eliminates seminal plasma and decreases the number of spermatozoa. Studies have shown that spermatozoa benefit if some of the seminal plasma is removed (Love et al, 2005, Braun et al, 1994, Kareskoski et al, 2006, MacPherson et al, 2002, Sieme et al, 2004, Waite et al, 2008). The EEF-study was carried out during June 2008 at the national stud at Flyinge, Sweden. Twelve stallions were included in the project, each stallion provided with four or five ejaculates. Aliquots were extended with two different types of extenders; Kenney´s or INRA96, and were analyzed for subjective, objective and progressive motility in fresh and cooled semen before and after selection by centrifugation. Semen was analyzed directly after collection and at 24 and 48 hours after collection. Between the analyses the semen was stored in 5°C. Assessment of motility was made both subjectively by phase contrast microscope and objectively with QualispermTM. The results from QualispermTM also included progressive motility and velocity. The results including selection/non selection were statistically analyzed with SAS (Statistical Analysis Systems Package, version 9, 1 SAS Institute Inc., Cary, N.C., USA, 1989) and both Correlation Procedure and Mixed Procedure were used. The parameters have then been compared with fertility results. Results from both subjective and objective assessment of motility shows that spermatozoa treated with INRA96 extender have higher motility both initially and 24 hours after collection than spermatozoa treated with Kenney´s. The subjective motility assessment also shows that semen extended with INRA96 has a higher motility after 48 hours than semen extended with Kenney´s. The results after selection are consistent with those above; spermatozoa extended with INRA96 have higher motility than spermatozoa treated with Kenney´s. The analysis shows that there was high significant correlation between the subjective and objective assessment methods. Furthermore the results implicate that spermatozoa after centrifugation maintain their motility longer, both with INRA96 and Kenney´s, than spermatozoa that are not selected. Results from the study also suggest that there is a correlation between sperm velocity and pregnancy results. High velocity of spermatozoa yields high percentage of pregnancy. These results agree with results from earlier studies (2005, 2006). Furthermore a comparison between pregnancy results and subjective and objective method of analysis show that the subjective method, in this particular study, is more reliable. To sum up: QualispermTM is an objective method of analysis that need certain adjustments. There still is a subjective element to the method and with consideration of the amount of time the analysis take, the amount of training required, and the sources of error all the above mentioned creates, the method is not usable in practice today. Nevertheless QualispermTM has several advantages to traditional subjective assessment method since the program can analyze more parameters. A further development of QualispermTM could very well increase the applicability of the method for semen analysis in the field