Durchflusszytometrische Verfahren zur Beurteilung der Spermaqualität nach einer experimentell induzierten Hyperthermie am Bullenhoden
Beschreibung
vor 19 Jahren
The aim of this study was to examine if the assessments of sperm
quality can be better objectified and standardized by using flow
cytometric examinations. For this investigation temporary
deterioration of sperm quality was induced by a local heating of
testes in bulls. The scrotal surface temperature of 4 bulls was
increased during the local heating of testis by enclosing the
entire scrotum for 48 hours by 6.4°C to 7.1°C. Semen was collected
3 times a week from Day -7 to Day 61 and once a week from Day 68 to
Day 83 (Day 0 = the day of scrotal insulation). Each ejaculate was
conventionally examined (numbers of total spermatozoa, sperm
concentration, morphology and proportion of vital spermatozoa after
staining with bromphenol blue nigrosin). The percentage of motile
spermatozoa and progressive motile spermatozoa of motile sperm were
determined with a computer-assisted motility analysis. Using flow
cytometry the proportions of sperm with the following parameters
were examined and analyzed: intact plasma membranes after staining
with SYBR14/PI, high mitochondrial membrane potentials after
staining with JC1, damaged acrosome status after staining with
FITC-PNA/SYTO17/PI, and sperm with defective chromatin structure or
rather with high DNA fragmentation index (DFI) by SCSA. The
deterioration of sperm quality after elevating testicular
temperature was correspondent to a large extent to the typical
changes of the conventional sperm parameters, which result is in
agreement with previous similar studies. After testicular
hyperthermia (Day 0) changes of sperm quality occurred in the
following sequence. There was a notable increase in secondary sperm
abnormalities as well as a decrease in sperm motility on Day 9
after testicular hyperthermia. The proportions of vital spermatozoa
after staining with bromphenol blue nigrosin, total sperm count and
sperm concentration were decreased on Day 12 for the first time. At
the same time the primary sperm abnormalities began to increase.
The primary abnormalities most frequently encountered were
morphological head defects of spermatozoa. Concurrent with the
changes specified above the following alterations of sperm
parameters were observed with flow cytometry. Beginning on Day 7
after testicular hyperthermia sperm with defective acrosome status
increased. From Day 9 on sperm with intact plasma membranes and
sperm with high mitochondrial membrane potentials began to
decrease. On Day 12 after testicular hyperthermia the proportion of
sperm with defective chromatin structure (spermatozoa with high
DFI) started to increase significantly. The relationships between
the proportions of defective sperm chromatin structure assessed
with the SCSATM test and the proportions of sperm head defects were
highly significant (r = 0.81; P < 0.0001). The proportion of
sperm with a high mitochondrial membrane potential correlated
positively to the sperm motility (r = 0.83; P < 0.0001).
Significant correlations between the viability assessed by light
microscopy and the percentages of spermatozoa with intact plasma
membrane obtained by flow cytometry (r = 0.77; P < 0.0001)
occurred. The increase of the proportion of the sperm with
defective chromatin structure (spermatozoa with high DFI) was more
clearly pronounced than that of the morphological abnormal sperm
heads. This result indicates that heat stress had led to chromosome
defects not only in morphologically abnormal but also in normally
appearing sperm cells. In addition, morphological sperm
abnormalities, especially sperm head defects, may be indicative of
chromosome abnormalities also in normally appearing sperm cells of
an ejaculate. This study shows that flow cytometric assessments of
the ejaculate is a reliable and objective method in semen
investigation. It provides additional important information about
the sperm quality. In further studies, it has to be clarified,
whether the prospective fertilization capacity from an ejaculate
and/or the fertility of a bull can be predicted with this method
more reliable than with the conventional sperm evaluation.
quality can be better objectified and standardized by using flow
cytometric examinations. For this investigation temporary
deterioration of sperm quality was induced by a local heating of
testes in bulls. The scrotal surface temperature of 4 bulls was
increased during the local heating of testis by enclosing the
entire scrotum for 48 hours by 6.4°C to 7.1°C. Semen was collected
3 times a week from Day -7 to Day 61 and once a week from Day 68 to
Day 83 (Day 0 = the day of scrotal insulation). Each ejaculate was
conventionally examined (numbers of total spermatozoa, sperm
concentration, morphology and proportion of vital spermatozoa after
staining with bromphenol blue nigrosin). The percentage of motile
spermatozoa and progressive motile spermatozoa of motile sperm were
determined with a computer-assisted motility analysis. Using flow
cytometry the proportions of sperm with the following parameters
were examined and analyzed: intact plasma membranes after staining
with SYBR14/PI, high mitochondrial membrane potentials after
staining with JC1, damaged acrosome status after staining with
FITC-PNA/SYTO17/PI, and sperm with defective chromatin structure or
rather with high DNA fragmentation index (DFI) by SCSA. The
deterioration of sperm quality after elevating testicular
temperature was correspondent to a large extent to the typical
changes of the conventional sperm parameters, which result is in
agreement with previous similar studies. After testicular
hyperthermia (Day 0) changes of sperm quality occurred in the
following sequence. There was a notable increase in secondary sperm
abnormalities as well as a decrease in sperm motility on Day 9
after testicular hyperthermia. The proportions of vital spermatozoa
after staining with bromphenol blue nigrosin, total sperm count and
sperm concentration were decreased on Day 12 for the first time. At
the same time the primary sperm abnormalities began to increase.
The primary abnormalities most frequently encountered were
morphological head defects of spermatozoa. Concurrent with the
changes specified above the following alterations of sperm
parameters were observed with flow cytometry. Beginning on Day 7
after testicular hyperthermia sperm with defective acrosome status
increased. From Day 9 on sperm with intact plasma membranes and
sperm with high mitochondrial membrane potentials began to
decrease. On Day 12 after testicular hyperthermia the proportion of
sperm with defective chromatin structure (spermatozoa with high
DFI) started to increase significantly. The relationships between
the proportions of defective sperm chromatin structure assessed
with the SCSATM test and the proportions of sperm head defects were
highly significant (r = 0.81; P < 0.0001). The proportion of
sperm with a high mitochondrial membrane potential correlated
positively to the sperm motility (r = 0.83; P < 0.0001).
Significant correlations between the viability assessed by light
microscopy and the percentages of spermatozoa with intact plasma
membrane obtained by flow cytometry (r = 0.77; P < 0.0001)
occurred. The increase of the proportion of the sperm with
defective chromatin structure (spermatozoa with high DFI) was more
clearly pronounced than that of the morphological abnormal sperm
heads. This result indicates that heat stress had led to chromosome
defects not only in morphologically abnormal but also in normally
appearing sperm cells. In addition, morphological sperm
abnormalities, especially sperm head defects, may be indicative of
chromosome abnormalities also in normally appearing sperm cells of
an ejaculate. This study shows that flow cytometric assessments of
the ejaculate is a reliable and objective method in semen
investigation. It provides additional important information about
the sperm quality. In further studies, it has to be clarified,
whether the prospective fertilization capacity from an ejaculate
and/or the fertility of a bull can be predicted with this method
more reliable than with the conventional sperm evaluation.
Weitere Episoden
Kommentare (0)