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.