Evaluierung durchflusszytometrischer Verfahren zur Beurteilung der Qualität von kryokonserviertem Hengstsperma

The aim of these examinations was to check various flow cytometric methods to establish whether they are suitable objective methods for assessing the quality of cryopreserved sperm from stallions. The plasma membrane integrity, the mitochondrial membrane potential, the acrosomal status and the integrity of the chromatin structure in cryopreserved sperm from stallions were assessed. For this purpose various fluorescent stainings were carried out both immediately after the semen samples were thawed and after a three-hour period of incubation at 37 C with SYBR® 14, JC-1 and SYTO® 17 / propidium iodide / FITC-PNA and a sperm chromatin structure analysis (SCSA™) and were then evaluated. The sperm quality parameters obtained by flow cytometry showed good reproducibility. The results of measurements of three different straws of an ejaculate which were carried out independently of each other did not differ significantly. The intra-class correlation coefficients were between 0.88 and 0.98. The flow cytometric tests evaluated in this examination were therefore considered to be reliable. In order to demonstrate the correlations between the routine examination methods for assessing the sperm quality and the analysis processes evaluated in this study, the viability and the morphology of the sperms after thawing were assessed by light microscopy using bromide phenol blue nigrosine smears. A computer controlled motility analysis was also carried out. Moderate correlations (0.60 > r > 0.58; p < 0.0001) were obtained between the viability assessed by light microscopy and the percentage of vital sperms obtained by flow cytometry. The proportion of progressively forward moving sperms correlated positively (r = 0.50; p < 0.01) to the proportion of sperms with a high mitochondrial membrane potential. The relationships between the results of the SCSA™ test and the proportions of both primary and secondary sperm anomalies were weakly pronounced (r = 0.24; p < 0.01). In the flow cytometric assessment of the viability, the mitochondrial membrane potential and the integrity of the acrosome it was shown that the ejaculate-related variations were approximately the same as the variations between the stallions. Regarding the results of the sperm chromatin structure the individual variations (88 - 91%) were considerably greater than those dependent on the ejaculate (9 – 12%). It was also shown that the results of the flow cytometric sperm quality assessment did not vary significantly in stallions which had a period (four months) of sexual rest before providing ejaculate compared to stallions which ejaculates were collected with a regular frequency. In terms of the animals’ age there was only a weak relationship (r = 0.43; p < 0.01) with integrity of the sperm chromatin structure. The results of the flow cytometric methods for assessing the viability (SYBR® 14/PI, JC-1 and SYTO® 17/FITC-PNA/PI combination staining) were comparable (r > 0.96; p < 0.0001). The latter is the only one of these tests which provides additional information about the integrity of the acrosome of the sperms. The combination of this triple staining with the sperm chromatin structure analysis (SCSA™) also provides further important information about the sperm quality. To check the relationships between fertility of the stallions and the various sperm quality parameters obtained by flow cytometry, only seasonal pregnancy rates from fresh semen insemination were available. In terms of the viability and the acrosomal status of the sperms after thawing, these showed no correlations (p > 0.05) with the fertility of the stallions. On the other hand, a negative relationship was established between the integrity of the chromatin structure and the fertility of the stallions (r = - 0.51 / - 0.59). This study shows that the flow cytometric assessment of the quality of cryopreserved sperm from stallions represents a reliable and objective method. In addition to the routine assessment of sperm quality, it provides important additional information, for example about the intactness of the acrosome and the integrity of the sperm chromatin structure.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2003

Author dc:creator

• Krienke, Matthias

Repository record source_url

https://edoc.ub.uni-muenchen.de/1338/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:1338